Low sequence bias single-stranded DNA ligation

ABSTRACT

The invention provides compositions and methods for ligating single stranded nucleic acids wherein the ligation is based on fast, efficient, and low-sequence bias hybridization of an acceptor molecule with a donor molecule. In one embodiment, the structure of the donor molecule comprises a stem-loop intramolecular nucleotide base pairing (i.e., hairpin) and a 3′-overhang region such that the overhang is able to hybridize to nucleotides present in the 3′ end of the acceptor molecule.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Ser.No. 61/750,469, filed Jan. 9, 2013, the contents of which areincorporated by reference herein in their entirety.

BACKGROUND OF THE INVENTION

Intermolecular single-stranded DNA (ssDNA) ligation is important forvarious biotechnical applications, such as LMPCR (Zhang et al., 1996,Nucleic Acids Res. 24:990-991; Dai et al., 2000, Nat. Biotech.18:1108-1111; Yeku and Frohman, 2011, Methods Mol. Biol. 703:107-122)and cDNA library construction (Levin et al., 2010, Nat. Meth. 7:709-715;Lucks et al., 2011, Proc. Natl. Acad. Sci. USA 108:11063-11068), each ofwhich require a fixed sequence DNA oligonucleotide to ligate to anunknown 3′-end of a cDNA. Currently, only a few protocols are availableto perform such intermolecular ssDNA ligations, which use Circligase I(Lucks et al., 2011, Proc. Natl. Acad. Sci. USA 108:11063-11068; Li etal., 2006, Anal. Biochem. 349:242-246; Blondal et al., 2005, NucleicAcids Res. 33:135-142) or T4 RNA ligase I (Zhang et al., 1996, NucleicAcids Res. 24:990-991; Tessier et al., 1986, Anal. Biochem.158:171-178). In addition, Circligase II recently became commerciallyavailable; however Circligase II is identical to Circligase I, differingonly in the level of protein adenylation. The nucleotide preferences,referring to the likelihood of ligation to a certain base (given anequal concentration of the 4 bases in the reaction mixture), of theseligation methods on intermolecular ssDNA ligations, however, were notavailable prior to the present invention.

Identification and remediation of nucleotide bias in ssDNA ligation iscrucial because such bias can fail to quantitatively capture theoriginal information stored in the DNA sample. Indeed, nucleotidepreference in nucleic acid ligations can potentially lead tomisinterpretation of gene expression levels (Jayaprakash et al., 2011,Nucleic Acids Res. 39(21):e141; McCormick et al., 2011, Silence 2(1):2;Linsen, et al., 2009, Nat. Meth. 6:474-476). Nucleotide bias andinefficiencies of ssDNA ligation hampers ligation methods currently inuse.

Thus, there is a need in the art for compositions and methods providingfast, efficient, and low-sequence bias ligation of ssDNAs. The presentinvention satisfies this unmet need.

SUMMARY OF THE INVENTION

The invention provides a method of producing a ligated single strandednucleic acid molecule. In one embodiment, the method comprises: a)contacting a single-stranded acceptor nucleic acid molecule with a donornucleic acid molecule, wherein the donor nucleic acid molecule comprisesone or more nucleic acids having a stem region and a single-stranded 3′terminal overhang region; b) hybridizing the single stranded 3′ terminaloverhang region of the donor nucleic acid molecule to the acceptormolecule thereby forming an acceptor-donor hybrid molecule comprising anick or gap between the acceptor nucleic acid and donor nucleic acidmolecule; c) and ligating the 5′ end of the donor nucleic acid moleculeto the 3′ end of the acceptor nucleic acid molecule thereby generating aligated product.

In one embodiment, the ligation step is accomplished after thehybridization, wherein the hybridization step positions the acceptor anddonor molecule in a way that ligation occurs under conditions that allowfor ligation between the 5′ end of the donor nucleic acid molecule andthe 3′ end of the acceptor nucleic acid molecule.

In one embodiment, the ligation occurs by enzymatic means. In anotherembodiment, the enzymatic means comprises using DNA ligase. In yetanother embodiment, the DNA ligase is T4 DNA ligase.

In one embodiment, the ligation occurs by chemical means.

In one embodiment, the 3′ terminal overhang region of donor moleculecomprises at least 1 bases.

In one embodiment, the stem region of the donor molecule is doublestranded and comprises at least 4 nucleotide pairs. In anotherembodiment, the stem region comprises at least one mismatched pair.

In one embodiment, the donor molecule further comprises a loopstructure, wherein the loop structure comprises at least 2 bases. Inanother embodiment, the loop structure comprises a portion of a primerbinding site.

The invention also provides a composition comprising a donor nucleicacid molecule, wherein the molecule comprises a stem-loop structure anda 3′ overhang, further wherein the molecule comprises a continuousprimer binding site that encompasses a portion of the stem and a portionof the loop structure.

In one embodiment, the stem portion of the stem-loop structure comprisesat least 3 nucleotide base pairs and at least one mismatch pair.

In one embodiment, the 3′ overhang comprises at least 4 nucleotides.

In one embodiment, the primer binding site comprises 8 nucleotides.

In one embodiment, the donor molecule is hybridized to a single strandednucleic acid acceptor molecule to form a hybridized molecule comprisingthe donor molecule and the acceptor molecule.

In one embodiment, the hybridized molecule is stable at a temperature ashigh as 65° C.

The invention also provides a kit comprising a donor nucleic acidmolecule. In one embodiment, the molecule comprises a stem-loopstructure and a 3′ overhang, further wherein the molecule comprises acontinuous primer binding site that encompasses a portion of the stemand a portion of the loop structure.

In one embodiment, the kit comprises a DNA ligase. In one embodiment,the DNA ligase is T4 DNA ligase.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of preferred embodiments of theinvention will be better understood when read in conjunction with theappended drawings. For the purpose of illustrating the invention, thereare shown in the drawings embodiments which are presently preferred. Itshould be understood, however, that the invention is not limited to theprecise arrangements and instrumentalities of the embodiments shown inthe drawings.

FIG. 1, comprising FIG. 1A through FIG. 1D, depicts the results ofexperiments examining ssDNA ligation by different ligases. FIG. 1Adepicts the design of the ssDNA ligation assay. Mfold-predictedsecondary structure of the acceptor and donor. FIG. 1B through FIG. 1Ddepict the nucleotide preference of ligations performed using CircligaseI, T4 RNA ligase I, and initial tests of T4 DNA ligase, respectively.FIG. 1B and FIG. 1C represent prior methods of ssDNA ligation, whileFIG. 1D is the initial version of the method developed herein. Laneslabeled ‘A’, ‘G’, ‘C’, and ‘T’ refer to the identity of the 3′ endnucleotide of the acceptor, which differs in each lane as indicated.Samples were fractionated on 10% urea-polyacrylamide gels and subjectedto PhosphorImager scanning Reaction conditions are described in detailelsewhere herein. Below each gel, the proposed mode of reaction of thecorresponding ligase is provided; note that only the method depicted inFIG. 1D is template-mediated.

FIG. 2 depicts a ligation model for ssDNA ligation using T4 DNA ligaseand positions and identities of DNA mutations. These reactions were in20% PEG 8000, 0.5 M betaine, 100:1 donor:acceptor ratio, and 15 U T4 DNAligase for 12 h at 16° C. SEQ ID NO: 28 represents the depicted portionof the donor molecule. The mini-hairpin (nucleotides 22-33) of the donorsequence is shown in FIG. 1A. Although base pairs 6:12 and 7:11 shown onFIG. 2 (dotted) were not predicted by Mfold using the WT donor (FIG.1A), they were predicted to form when base pair 4:14 was changed to WCbase pair (M13-M15). In addition, it was reasoned that under conditionswhere 20% PEG was added, these base pairs may form. For gels showingligation results of M1-M18, see FIGS. 16-21.

FIG. 3, comprising FIG. 3A through FIG. 3C, depicts the results ofexperiments demonstrating ssDNA ligation and kinetics. FIG. 3A depictsthe design of nucleotide preference ligation using the optimized donor(40 mer) that contains a random hexamer templating region (N6) and anacceptor (24 mer). SEQ ID NO: 29 represents the depicted portion of thedonor molecule. The 7 nt SapI/BspQI site is in bold. FIG. 3B is an imagedepicting the ssDNA ligation result using the optimized donor from panel3A. Lane labels ‘A’, ‘G’, ‘C’, and ‘T’ refer to the DNA base of the 3′end nucleotide of the acceptor. The % yields reported are the average ofthree trials, with standard deviation indicated. FIG. 3C is a graphdepicting the kinetics of nucleotide preference for ssDNA ligation. Datapoints are the average of three trials, and error bars are standarddeviations.

FIG. 4 is an image depicting the results of experiments examining ssDNAligation of optimized donor (40 mer) and 3 additional FAM labeledacceptors (16, 19, 21 mer) using T4 DNA ligase. Reactions were in 20%PEG 8000, 0.5 M betaine, 100:1 donor:acceptor ratio, and 15 U T4 DNAligase for 12 h at 16° C. Samples were fractionated on a 10%urea-polyacrylamide gel and subjected to PhosphorImager scanning.

FIG. 5, comprising FIG. 5A and FIG. 5B, depicts the results ofexperiments examining ligation-mediated PCR using the optimized hairpindonor construct and a 91 nucleotide acceptor, followed by SapI/BspQIrestriction digestion. FIG. 5A depicts the results of experiments wherethe ssDNA ligation was performed as described in FIG. 4, except that 1pmol of 91 mer acceptor was used and the reaction was conducted for 0.5h. The control lanes did not have T4 DNA ligase added. Thirty rounds ofPCR were carried out using New England Biolabs Taq DNA polymerase withvarying annealing temperatures as indicated. The PCR primers werecomplementary to the 5′-end of the acceptor and the fixed region of thedonor. SapI or BspQI digestion was performed on the 131 bp PCR productusing NEB buffer 4 under the vendor's recommended condition. Thedigestion was completed and a product of 87/91 bp was observed after 1 hreaction for both enzymes. Shown is an ethidium bromide-stained agarosegel. FIG. 5B depicts a general scheme for LMPCR and restriction digest.SEQ ID NO: 29 represents the depicted portion of the donor molecule.

FIG. 6 is an image depicting the results of a nucleotide preference testof donor oligonucleotides 2 and 3 using Circligase I. Donor sequencesare provided in FIG. 23. Donor 2 has minimal secondary structure, andDonor 3 has 3 randomized nucleotides at its 5′-end. The reactioncontained 100 pmol of 5′p donor, 1 pmol of Cy5-labelled acceptor, 50 mMMOPS (pH 7.5), 10 mM KCl, 5 mM MgCl₂, 1 mM DTT, 0.05 mM ATP, 2.5 mMMnCl₂ and 200 U Circligase I. Reaction was performed at 65° C. for 12 h,and then 85° C. for 15 min to deactivate the enzyme.

FIG. 7 is a set of images depicting the results of experiments examiningthe ssDNA ligation kinetics and nucleotide preference of wild-type (WT;33 mer) donor oligonucleotide using Circligase I. The reaction contained100 pmol of 5′p donor, 1 pmol of Cy5-labelled acceptor, 20% PEG 8000, 50mM MOPS (pH 7.5), 10 mM KCl, 5 mM MgCl₂, 1 mM DTT, 0.05 mM ATP, 2.5 mMMnCl₂ and 200 U Circligase I. Reaction was performed at 68° C. and timepoints were taken at indicated time intervals.

FIG. 8 is a graph depicting the results of a nucleotide preference testof donor oligonucleotide 3 under different donor:acceptor ratios usingCircligase I. The reaction contained 100 or 300 pmol of 5′p donor, 1pmol of Cy5-labelled acceptor, 50 mM MOPS (pH 7.5), 10 mM KCl, 5 mMMgCl₂, 1 mM DTT, 0.05 mM ATP, 2.5 mM MnCl₂ and 200 U Circligase I.Reaction was performed at 65° C. for 12 h, and then 85° C. for 15 min todeactivate the enzyme. The fraction ligated shown is the average valueof 3 trials and the error bar is standard deviation. Note thatincreasing 3× in donor:acceptor ratio did not alter the fraction ligatedsignificantly, indicating that the donor:acceptor ratio is saturating at100:1 ratio.

FIG. 9 is an image depicting the results of ssDNA ligation of WT donorand “G” acceptor using either New England Biolabs (NEB) or a laboratorypreparation of T4 DNA ligase. 12 U of NEB T4 DNA ligase and 15 U oflaboratory preparation T4 DNA ligase were used, and the ligationreaction contained 100 pmol of 5′p donor, 1 pmol of Cy5-labelledacceptor, 50 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 10 mM DTT, and 1 mM ATP.Reaction was performed at 16° C. for 12 h. Results are quantified in “%yield/unit” to allow comparison between these two ligases.

FIG. 10 is an image depicting the results of ssDNA ligation controlexperiments of WT donor and “G” acceptor using T4 DNA ligase. Individualcomponents were omitted from the standard ligation reaction asindicated. The reaction was performed at 16° C. for 12 h. ‘NA’=notapplicable as no fluorophore was present.

FIG. 11 is an image depicting the results of ssDNA ligation of WT donorand “G” acceptor under different temperatures using T4 DNA ligase. Thestandard ligation reaction containing 100 pmol of 5′p donor, 1 pmol ofCy5-labelled acceptor, 50 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 10 mM DTT,1 mM ATP, 15 U laboratory preparation of T4 DNA ligase was incubated for12 h at different temperatures as indicated.

FIG. 12 is an image depicting the results of ssDNA ligation of WT donorand “G” acceptor under different PEG 8000 concentrations using T4 DNAligase. The standard ligation reaction containing 100 pmol of 5′p donor,1 pmol of Cy5-labelled acceptor, 50 mM Tris-HCl (pH 7.5), 10 mM MgCl²,10 mM DTT, 1 mM ATP, 15 U laboratory preparation of T4 DNA ligase wasincubated for 12 h at 16° C. PEG 8000 concentration was varied asindicated.

FIG. 13 is an image depicting the results of ssDNA ligation of WT donorand “G” acceptor under different betaine concentrations using T4 DNAligase. The standard ligation reaction containing 100 pmol of 5′p donor,1 pmol of Cy5-labelled acceptor, 50 mM Tris-HCl (pH 7.5), 10 mM MgCl²,10 mM DTT, 1 mM ATP, 15 U laboratory preparation of T4 DNA ligase wasincubated for 12 h at 16° C. Betaine concentration was varied asindicated. The choice of 0.5 M betaine for optimized reaction was basedon the bell-shape response observed here.

FIG. 14 is an image depicting the results of ssDNA ligation kinetics ofWT donor and “G” acceptor under different donor:acceptor ratios using T4DNA ligase. The standard ligation reaction containing different ratiosof 5′p donor and Cy5-labelled acceptor as indicated, 50 mM Tris-HCl (pH7.5), 10 mM MgCl₂, 10 mM DTT, 1 mM ATP, 15 U laboratory preparation ofT4 DNA ligase was incubated for 12 h at 16° C.

FIG. 15, comprising FIG. 15A through FIG. 15C, depicts the results ofssDNA ligation kinetics of WT donor and “G” acceptor under standard andoptimized conditions using T4 DNA ligase. FIG. 15A is an image depictingthe ligation time course for standard conditions. FIG. 15B is an imagedepicting the ligation time course for optimized conditions. FIG. 15C isa ssDNA ligation kinetics plot. Standard ligation conditions contained100 pmol of 5′p donor, 1 pmol of Cy5-labelled acceptor, 50 mM Tris-HCl(pH 7.5), 10 mM MgCl₂, 10 mM DTT, 1 mM ATP, and 15 U laboratorypreparation of T4 DNA ligase at 16° C. Optimized ligation conditionsadded 20% PEG 8000 and 0.5 M betaine.

FIG. 16 is an image depicting the results of ssDNA ligation of M1-M3,M16 and WT using T4 DNA ligase. The ligation reaction contained 100 pmolof 5′p donor, 1 pmol of Cy5-labelled acceptor, 50 mM Tris-HCl (pH 7.5),10 mM MgCl₂, 10 mM DTT, 1 mM ATP, 20% PEG 8000 and 0.5M betaine. Thereaction was performed at 16° C. for 12 h, and then 65° C. for 15 min todeactivate the enzyme.

FIG. 17 is an image depicting the results of ssDNA ligation of M4-M7using T4 DNA ligase. The ligation reaction contained 100 pmol of 5′pdonor, 1 pmol of Cy5-labelled acceptor, 50 mM Tris-HCl (pH 7.5), 10 mMMgCl₂, 10 mM DTT, 1 mM ATP, 20% PEG 8000 and 0.5M betaine. The reactionwas performed at 16° C. for 12 h, and then 65° C. for 15 min todeactivate the enzyme.

FIG. 18 is an image depicting the results of ssDNA ligation of M8-M10and WT using T4 DNA ligase. The ligation reaction contained 100 pmol of5′p donor, 1 pmol of Cy5-labelled acceptor, 50 mM Tris-HCl (pH 7.5), 10mM MgCl₂, 10 mM DTT, 1 mM ATP, 20% PEG 8000 and 0.5M betaine. Thereaction was performed at 16° C. for 12 h and then 65° C. for 15 min todeactivate the enzyme. The 1 min samples were loaded and ran slightlybefore the 12 h samples.

FIG. 19 is an image depicting the results of ssDNA ligation of M11-M12using T4 DNA ligase. The ligation reaction contained 100 pmol of 5′pdonor, 1 pmol of Cy5-labelled acceptor, 50 mM Tris-HCl (pH 7.5), 10 mMMgCl₂, 10 mM DTT, 1 mM ATP, 20% PEG 8000 and 0.5 M betaine. The reactionwas performed at 16° C. for 12 h, and then 65° C. for 15 min todeactivate the enzyme.

FIG. 20 is an image depicting the results of ssDNA ligation of M13-15using T4 DNA ligase. The ligation reaction contained 100 pmol of 5′pdonor, 1 pmol of Cy5-labelled acceptor, 50 mM Tris-HCl (pH 7.5), 10 mMMgCl₂, 10 mM DTT, 1 mM ATP, 20% PEG 8000 and 0.5 M betaine. The reactionwas performed at 16° C. for 12 h, and then 65° C. for 15 min todeactivate the enzyme.

FIG. 21 is an image depicting the results of ssDNA ligation of M17 andM18 using T4 DNA ligase. The ligation reaction contained 100 pmol of 5′pdonor, 1 pmol of Cy5-labelled acceptor, 50 mM Tris-HCl (pH 7.5), 10 mMMgCl₂, 10 mM DTT, 1 mM ATP, 20% PEG 8000 and 0.5 M betaine. The reactionwas performed at 16° C. for 12 h and then 65° C. for 15 min todeactivate the enzyme.

FIG. 22 is an image depicting the results of experiments examining ssDNAligation kinetics and nucleotide preference of optimized construct usingT4 DNA ligase. The ligation reaction contained 400 pmol of 5′p donor, 1pmol of Cy5-labelled acceptor, 50 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 10mM DTT, 1 mM ATP, 20% PEG 8000 and 0.5 M betaine. The reaction wasperformed at 16° C. 400 p mol of 5′p donor was used since only ¼ of thedonor molecules had the complementary nucleotide that matched the 3′ endnucleotide of the acceptor.

FIG. 23 depicts the DNA sequence information for constructs used herein.

FIG. 24, comprising FIGS. 24A through 24C, is a schematic of a generaldesign for the acceptor, donor, and hybrid molecule of the invention.FIG. 24A depicts a schematic of the acceptor molecule whereby thenucleic acid molecule can comprise any base and be of any length. FIG.24B depicts a schematic of the donor molecule having a stem structure, aloop region, and a 3′-overhang. FIG. 24C depicts a schematic of theunligated hybrid molecule comprising a nick. The hybrid moleculecomprises a larger and more stable stem structure than was present inthe donor alone.

FIG. 25 depicts a flow chart for the targeted determination of RNAstructure for high- and low-abundance RNAs. Either DMS/SHAPE-RT (Steps1-3) or DMS/SHAPE-LMPCR can be used (all steps). In the first step,total RNA is treated with DMS or SHAPE reagent, either in vitro or invivo. In DMS/SHAPE-RT, a radiolabeled 5′-³²P gene-specific primer isused for the RT step, whereas in DMS/SHAPE-LMPCR, an unlabelled 5′-OHgene-specific primer is used for the RT step. Next, the RNA is degradedby base hydrolysis. For DMS/SHAPE-LMPCR, the unlabelled cDNA generatedfrom RT is ligated to a DNA adaptor by single-stranded (ss) DNAligation. Subsequently, a 5′-OH DNA adaptor-specific forward primer anda radioactive 5′-³²P (for PAGE) or a 5′-FAM (for CE) gene-specificnested reverse primer are used for PCR amplification of the ligated cDNAfragments. For the (−)DMS/SHAPE control reaction, all steps are the sameexcept that DMS/SHAPE treatment is omitted.

FIG. 26 depicts the results of an experiment comparing DMS/SHAPE-RT withDMS/SHAPE-LMPCR on Arabidopsis thaliana 5.8S rRNA using either T4 DNAligase or Circligase I. Rectangles depict new bands observed only by T4DNA ligase method (left panel, lane 8) and not by existing Circligasemethod (left panel, lane 10). Compare lanes 8 and 10 (left panel) to thelow sensitivity method of DMS-RT (left panel, lane 6). Asterisks denotebands in lane 6 (left) and where corresponding LMPCR band should be inlanes 8 and 10 (left), (missing for some bands in lane 10). The gel onthe right panel is a technical replicate for DMS/SHAPE-RT andDMS/SHAPE-LMPCR (using T4 DNA ligase) on 5.8S rRNA.

FIG. 27 depicts an overview of structure-seq. Arabidopsis seedlings aretreated with DMS. Reverse transcription is performed using randomhexamers (N₆) with adaptors (thicker black lines). Reverse transcriptasestalls one nucleotide before DMS-modified As and Cs (black crosses).Single-stranded (ss) DNA ligation attaches a single-stranded DNA linker(thicker black line) to the 3′ end. Double-stranded DNA is generated byPCR. A (−)DMS library is prepared in parallel. Deep sequencing isperformed with different indices for (+)DMS and (−)DMS libraries. Countsof the reverse transcriptase (RT) stops are normalized and subtracted.Pie charts depict percentages of RNA types for the (+)DMS (left) and(−)DMS (right) libraries. The non-rRNA and non-mRNA slice of the pierepresent other RNA types plus unmappable reads

DETAILED DESCRIPTION

The present invention is based on the development of a rationalexperimental design to remove the inherent nucleotide bias andinefficiencies in ligation methods currently in use. The invention isbased on a novel hybridization-based strategy that allows for fast,efficient and low-sequence bias ligation of two single stranded DNAs(ssDNA). Accordingly, the invention provides a method of ligating singlestranded nucleic acids that overcomes the nucleotide bias andinefficiencies associated with currently used protocols.

In one embodiment, the method of the invention comprises a ligationapproach that is based on hybridization of at least two single strandednucleic acids, one of the single stranded nucleic acid is referredherein as an acceptor molecule and the other single stranded nucleicacids is referred herein as a donor molecule.

In some instances, the donor molecule comprises a hairpin structure. Inother instances, the hairpin donor molecule comprises a 3′-overhang. Inone embodiment, the ligation between the acceptor molecule and the donormolecule is accomplished through the actions of a ligase. In certainembodiments, the ligase is a T4 DNA ligase. Generally, the donormolecule hybridizes with an acceptor 3′-end to yield the desiredligation product (e.g., a hybrid molecule comprising the acceptor anddonor molecule).

The ability to ligate single stranded nucleic acids provides a valuabletechnique that can be applied to various protocols, including, thoseprotocols studying nucleic acids.

The ssDNA ligation composition and method of the present invention maybe used in a wide variety of protocols and technologies. For example, incertain embodiments, ssDNA ligation is used in the fields of molecularbiology, genomics, transcriptomics, epigenetics, nucleic acidsequencing, and the like. In one embodiment, ssDNA ligation may be usedin any technology that may require or benefit from the ligation ofssDNA.

In one embodiment, the ssDNA ligation composition and method of theinvention is applicable to DMS/SHAPE-LMPCR and Structure-Seq, andDMS-seq to obtain in vivo or in vitro RNA structural data at nucleotideresolution in low-abundance transcripts and genome-wide, respectively inany organism or tissue.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are described.

As used herein, each of the following terms has the meaning associatedwith it in this section.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

“About” as used herein when referring to a measurable value such as anamount, a temporal duration, and the like, is meant to encompassvariations of ±20% or ±10%, more preferably ±5%, even more preferably±1%, and still more preferably ±0.1% from the specified value, as suchvariations are appropriate to perform the disclosed methods.

“Amplification” refers to any means by which a polynucleotide sequenceis copied and thus expanded into a larger number of polynucleotidemolecules, e.g., by reverse transcription, polymerase chain reaction,and ligase chain reaction, among others. Amplification ofpolynucleotides encompasses a variety of chemical and enzymaticprocesses. The generation of multiple DNA copies from one or a fewcopies of a target or template DNA molecule during a polymerase chainreaction (PCR) or a ligase chain reaction (LCR) are forms ofamplification. Amplification is not limited to the strict duplication ofthe starting molecule. For example, the generation of multiple cDNAmolecules from a limited amount of RNA in a sample using reversetranscription (RT)-PCR is a form of amplification. Furthermore, thegeneration of multiple RNA molecules from a single DNA molecule duringthe process of transcription is also a form of amplification.

“Complementary” refers to the broad concept of sequence complementaritybetween regions of two nucleic acid strands or between two regions ofthe same nucleic acid strand. It is known that an adenine residue of afirst nucleic acid region is capable of forming specific hydrogen bonds(“base pairing”) with a residue of a second nucleic acid region which isantiparallel to the first region if the residue is thymine or uracil.Similarly, it is known that a cytosine residue of a first nucleic acidstrand is capable of base pairing with a residue of a second nucleicacid strand which is antiparallel to the first strand if the residue isguanine. A first region of a nucleic acid is complementary to a secondregion of the same or a different nucleic acid if, when the two regionsare arranged in an antiparallel fashion, at least one nucleotide residueof the first region is capable of base pairing with a residue of thesecond region. Preferably, the first region comprises a first portionand the second region comprises a second portion, whereby, when thefirst and second portions are arranged in an antiparallel fashion, atleast about 50%, and preferably at least about 75%, at least about 90%,or at least about 95% of the nucleotide residues of the first portionare capable of base pairing with nucleotide residues in the secondportion. More preferably, all nucleotide residues of the first portionare capable of base pairing with nucleotide residues in the secondportion.

“Encoding” refers to the inherent property of specific sequences ofnucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom. Thus, a gene encodes aprotein if transcription and translation of mRNA corresponding to thatgene produces the protein in a cell or other biological system. Both thecoding strand, the nucleotide sequence of which is identical to the mRNAsequence and is usually provided in sequence listings, and thenon-coding strand, used as the template for transcription of a gene orcDNA, can be referred to as encoding the protein or other product ofthat gene or cDNA. Unless otherwise specified, a “nucleotide sequenceencoding an amino acid sequence” includes all nucleotide sequences thatare degenerate versions of each other and that encode the same aminoacid sequence. Nucleotide sequences that encode proteins and RNA mayinclude introns.

As used herein, the term “fragment,” as applied to a nucleic acid,refers to a subsequence of a larger nucleic acid. A “fragment” of anucleic acid can be at least about 15 nucleotides in length; forexample, at least about 50 nucleotides to about 100 nucleotides; atleast about 100 to about 500 nucleotides, at least about 500 to about1000 nucleotides, at least about 1000 nucleotides to about 1500nucleotides; or about 1500 nucleotides to about 2500 nucleotides; orabout 2500 nucleotides (and any integer value in between).

“Homologous, homology” or “identical, identity” as used herein, refer tocomparisons among amino acid and nucleic acid sequences. When referringto nucleic acid molecules, “homology,” “identity,” or “percentidentical” refers to the percent of the nucleotides of the subjectnucleic acid sequence that have been matched to identical nucleotides bya sequence analysis program. Homology can be readily calculated by knownmethods. Nucleic acid sequences and amino acid sequences can be comparedusing computer programs that align the similar sequences of the nucleicor amino acids and thus define the differences. In preferredmethodologies, the BLAST programs (NCBI) and parameters used therein areemployed, and the ExPaSy is used to align sequence fragments of genomicDNA sequences. However, equivalent alignment assessments can be obtainedthrough the use of any standard alignment software.

As used herein, “homologous” refers to the subunit sequence similaritybetween two polymeric molecules, e.g., between two nucleic acidmolecules, e.g., two DNA molecules or two RNA molecules, or between twopolypeptide molecules. When a subunit position in both of the twomolecules is occupied by the same subunit, e.g., if a position in eachof two DNA molecules is occupied by adenine, then they are homologous atthat position. The homology between two sequences is a direct functionof the number of matching or homologous positions, e.g., if half (e.g.,five positions in a polymer ten subunits in length) of the positions intwo compound sequences are homologous then the two sequences are 50%homologous, if 90% of the positions, e.g., 9 of 10, are matched orhomologous, the two sequences share 90% homology. By way of example, theDNA sequences 5′ATTGCC 3′ and 5′TATGGC 3′ share 50% homology.

“Hybridization probes” are oligonucleotides capable of binding in abase-specific manner to a complementary strand of nucleic acid. Suchprobes include peptide nucleic acids, as described in Nielsen et al.,1991, Science 254, 1497-1500, and other nucleic acid analogs and nucleicacid mimetics. See U.S. Pat. No. 6,156,501.

The term “hybridization” refers to the process in which twosingle-stranded nucleic acids bind non-covalently to form adouble-stranded nucleic acid; triple-stranded hybridization is alsotheoretically possible. Complementary sequences in the nucleic acidspair with each other to form a double helix. The resultingdouble-stranded nucleic acid is a “hybrid.” Hybridization may bebetween, for example, two complementary or partially complementarysequences. The hybrid may have double-stranded regions and singlestranded regions. The hybrid may be, for example, DNA:DNA, RNA:DNA orDNA:RNA. Hybrids may also be formed between modified nucleic acids. Oneor both of the nucleic acids may be immobilized on a solid support.Hybridization techniques may be used to detect and isolate specificsequences, measure homology, or define other characteristics of one orboth strands.

The stability of a hybrid depends on a variety of factors including thelength of complementarity, the presence of mismatches within thecomplementary region, the temperature and the concentration of salt inthe reaction. Hybridizations are usually performed under stringentconditions, for example, at a salt concentration of no more than 1 M anda temperature of at least 25° C. For example, conditions of 5×SSPE (750mM NaCl, 50 mM Na Phosphate, 5 mM EDTA, pH 7.4) or 100 mM MES, 1 M Na,20 mM EDTA, 0.01% Tween-20 and a temperature of 25-50° C. are suitablefor allele-specific probe hybridizations. In a particularly preferredembodiment, hybridizations are performed at 40-50° C. Acetylated BSA andherring sperm DNA may be added to hybridization reactions. Hybridizationconditions suitable for microarrays are described in the Gene ExpressionTechnical Manual and the GeneChip Mapping Assay Manual available fromAffymetrix (Santa Clara, Calif.).

A first oligonucleotide anneals with a second oligonucleotide with “highstringency” if the two oligonucleotides anneal under conditions wherebyonly oligonucleotides which are at least about 75%, and preferably atleast about 90% or at least about 95%, complementary anneal with oneanother. The stringency of conditions used to anneal twooligonucleotides is a function of, among other factors, temperature,ionic strength of the annealing medium, the incubation period, thelength of the oligonucleotides, the G-C content of the oligonucleotides,and the expected degree of non-homology between the twooligonucleotides, if known. Methods of adjusting the stringency ofannealing conditions are known (see, e.g. Sambrook et al., 2012,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.).

As used herein, an “instructional material” includes a publication, arecording, a diagram, or any other medium of expression which can beused to communicate the usefulness of a compound, composition, vector,or delivery system of the invention in the kit for effecting alleviationof the various diseases or disorders recited herein. Optionally, oralternately, the instructional material can describe one or more methodsof alleviating the diseases or disorders in a cell or a tissue of amammal. The instructional material of the kit of the invention can, forexample, be affixed to a container which contains the identifiedcompound, composition, vector, or delivery system of the invention or beshipped together with a container which contains the identifiedcompound, composition, vector, or delivery system. Alternatively, theinstructional material can be shipped separately from the container withthe intention that the instructional material and the compound be usedcooperatively by the recipient.

As used herein, “isolate” refers to a nucleic acid obtained from anindividual, or from a sample obtained from an individual. The nucleicacid may be analyzed at any time after it is obtained (e.g., before orafter laboratory culture, before or after amplification.)

The term “label” as used herein refers to a luminescent label, a lightscattering label or a radioactive label. Fluorescent labels include, butare not limited to, the commercially available fluoresceinphosphoramidites such as Fluoreprime (Pharmacia), Fluoredite (Millipore)and FAM (ABI). See U.S. Pat. No. 6,287,778.

As used herein, the term “ligation agent” can comprise any number ofenzymatic or non-enzymatic reagents. For example, ligase is an enzymaticligation reagent that, under appropriate conditions, formsphosphodiester bonds between the 3′-OH and the 5′-phosphate of adjacentnucleotides in DNA molecules, RNA molecules, or hybrids. Temperaturesensitive ligases, include, but are not limited to, bacteriophage T4ligase and E. coli ligase. Thermostable ligases include, but are notlimited to, Afu ligase, Taq ligase, Tfl ligase, Tth ligase, Tth HB8ligase, Thermus species AK16D ligase and Pfu ligase (see for examplePublished P.C.T. Application WO00/26381, Wu et al., Gene, 76(2):245-254,(1989), Luo et al., Nucleic Acids Research, 24(15): 3071-3078 (1996).The skilled artisan will appreciate that any number of thermostableligases, including DNA ligases and RNA ligases, can be obtained fromthermophilic or hyperthermophilic organisms, for example, certainspecies of eubacteria and archaea; and that such ligases can be employedin the disclosed methods and kits. Further, reversibly inactivatedenzymes (see for example U.S. Pat. No. 5,773,258) can be employed insome embodiments of the present teachings. Chemical ligation agentsinclude, without limitation, activating, condensing, and reducingagents, such as carbodiimide, cyanogen bromide (BrCN), N-cyanoimidazole,imidazole, 1-methylimidazole/carbodiimide/cystamine, dithiothreitol(DTT) and ultraviolet light. Autoligation, i.e., spontaneous ligation inthe absence of a ligating agent, is also within the scope of theteachings herein. Detailed protocols for chemical ligation methods anddescriptions of appropriate reactive groups can be found in, among otherplaces, Xu et al., Nucleic Acid Res., 27:875-81 (1999); Gryaznov andLetsinger, Nucleic Acid Res. 21:1403-08 (1993); Gryaznov et al., NucleicAcid Res. 22:2366-69 (1994); Kanaya and Yanagawa, Biochemistry25:7423-30 (1986); Luebke and Dervan, Nucleic Acids Res. 20:3005-09(1992); Sievers and von Kiedrowski, Nature 369:221-24 (1994); Liu andTaylor, Nucleic Acids Res. 26:3300-04 (1999); Wang and Kool, NucleicAcids Res. 22:2326-33 (1994); Purmal et al., Nucleic Acids Res.20:3713-19 (1992); Ashley and Kushlan, Biochemistry 30:2927-33 (1991);Chu and Orgel, Nucleic Acids Res. 16:3671-91 (1988); Sokolova et al.,FEBS Letters 232:153-55 (1988); Naylor and Gilham, Biochemistry5:2722-28 (1966); and U.S. Pat. No. 5,476,930.

The term “mismatch,” “mismatch control” or “mismatch probe” refers to anucleic acid whose sequence is not perfectly complementary to aparticular target sequence. The mismatch may comprise one or more bases.While the mismatch(es) may be located anywhere in the mismatch probe,terminal mismatches are less desirable because a terminal mismatch isless likely to prevent hybridization of the target sequence. In aparticularly preferred embodiment, the mismatch is located at or nearthe center of the probe such that the mismatch is most likely todestabilize the duplex with the target sequence under the testhybridization conditions.

As used herein, the term “nucleic acid” refers to bothnaturally-occurring molecules such as DNA and RNA, but also variousderivatives and analogs. Generally, the probes, hairpin linkers, andtarget polynucleotides of the present teachings are nucleic acids, andtypically comprise DNA. Additional derivatives and analogs can beemployed as will be appreciated by one having ordinary skill in the art.

The term “nucleotide base”, as used herein, refers to a substituted orunsubstituted aromatic ring or rings. In certain embodiments, thearomatic ring or rings contain at least one nitrogen atom. In certainembodiments, the nucleotide base is capable of forming Watson-Crickand/or Hoogsteen hydrogen bonds with an appropriately complementarynucleotide base. Exemplary nucleotide bases and analogs thereof include,but are not limited to, naturally occurring nucleotide bases adenine,guanine, cytosine, 6 methyl-cytosine, uracil, thymine, and analogs ofthe naturally occurring nucleotide bases, e.g., 7-deazaadenine,7-deazaguanine, 7-deaza-8-azaguanine, 7-deaza-8-azaadenine, N6 delta2-isopentenyladenine (6iA), N6-delta 2-isopentenyl-2-methylthioadenine(2 ms6iA), N2-dimethylguanine (dmG), 7-methylguanine (7mG), inosine,nebularine, 2-aminopurine, 2-amino-6-chloropurine, 2,6-diaminopurine,hypoxanthine, pseudouridine, pseudocytosine, pseudoisocytosine,5-propynylcytosine, isocytosine, isoguanine, 7-deazaguanine,2-thiopyrimidine, 6-thioguanine, 4-thiothymine, 4-thiouracil,06-methylguanine, N6-methyladenine, 04-methylthymine,5,6-dihydrothymine, 5,6-dihydrouracil, pyrazolo[3,4-D]pyrimidines (see,e.g., U.S. Pat. Nos. 6,143,877 and 6,127,121 and PCT publishedapplication WO 01/38584), ethenoadenine, indoles such as nitroindole and4-methylindole, and pyrroles such as nitropyrrole. Certain exemplarynucleotide bases can be found, e.g., in Fasman, 1989, Practical Handbookof Biochemistry and Molecular Biology, pp. 385-394, CRC Press, BocaRaton, Fla., and the references cited therein.

The term “nucleotide”, as used herein, refers to a compound comprising anucleotide base linked to the C-1′ carbon of a sugar, such as ribose,arabinose, xylose, and pyranose, and sugar analogs thereof. The termnucleotide also encompasses nucleotide analogs. The sugar may besubstituted or unsubstituted. Substituted ribose sugars include, but arenot limited to, those riboses in which one or more of the carbon atoms,for example the 2′-carbon atom, is substituted with one or more of thesame or different Cl, F, —R, —OR, —NR2 or halogen groups, where each Ris independently H, C1-C6 alkyl or C5-C14 aryl. Exemplary ribosesinclude, but are not limited to, 2′-(C1-C6)alkoxyribose,2′-(C5-C14)aryloxyribose, 2′,3′-didehydroribose, 2′-deoxy-3′-haloribose,2′-deoxy-3′-fluororibose, 2′-deoxy-3′-chlororibose,2′-deoxy-3′-aminoribose, 2′-deoxy-3′-(C1-C6)alkylribose,2′-deoxy-3′-(C1-C6)alkoxyribose and 2′-deoxy-3′-(C5-C14)aryloxyribose,ribose, 2′-deoxyribose, 2′,3′-dideoxyribose, 2′-haloribose,2′-fluororibose, 2′-chlororibose, and 2′-alkylribose, e.g., 2′-O-methyl,4′-anomeric nucleotides, 1′-anomeric nucleotides, 2′-4′- and3′-4′-linked and other “locked” or “LNA”, bicyclic sugar modifications(see, e.g., PCT published application nos. WO 98/22489, WO 98/39352; andWO 99/14226). The term “nucleic acid” typically refers to largepolynucleotides.

The term “oligonucleotide” typically refers to short polynucleotides,generally, no greater than about 50 nucleotides. It will be understoodthat when a nucleotide sequence is represented by a DNA sequence (i.e.,A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C) inwhich “U” replaces “T.”

The term “polynucleotide” as used herein is defined as a chain ofnucleotides. Furthermore, nucleic acids are polymers of nucleotides.Thus, nucleic acids and polynucleotides as used herein areinterchangeable. One skilled in the art has the general knowledge thatnucleic acids are polynucleotides, which can be hydrolyzed into themonomeric “nucleotides.” The monomeric nucleotides can be hydrolyzedinto nucleosides. As used herein polynucleotides include, but are notlimited to, all nucleic acid sequences which are obtained by any meansavailable in the art, including, without limitation, recombinant means,i.e., the cloning of nucleic acid sequences from a recombinant libraryor a cell genome, using ordinary cloning and amplification technology,and the like, and by synthetic means. An “oligonucleotide” as usedherein refers to a short polynucleotide, typically less than 100 basesin length.

Conventional notation is used herein to describe polynucleotidesequences: the left-hand end of a single-stranded polynucleotidesequence is the 5′-end. The DNA strand having the same sequence as anmRNA is referred to as the “coding strand”; sequences on the DNA strandwhich are located 5′ to a reference point on the DNA are referred to as“upstream sequences”; sequences on the DNA strand which are 3′ to areference point on the DNA are referred to as “downstream sequences.” Inthe sequences described herein:

A=adenine,

G=guanine,

T=thymine,

C=cytosine,

U=uracil,

H=A, C or T/U,

R=A or G,

M=A or C,

K=G or T/U,

S=G or C,

Y=C or T/U,

W=A or T/U,

B=G or C or T/U,

D=A or G, or T/U,

V=A or G or C,

N=A or G or C or T/U.

The skilled artisan will understand that all nucleic acid sequences setforth herein throughout in their forward orientation, are also useful inthe compositions and methods of the invention in their reverseorientation, as well as in their forward and reverse complementaryorientation, and are described herein as well as if they were explicitlyset forth herein.

“Primer” refers to a polynucleotide that is capable of specificallyhybridizing to a designated polynucleotide template and providing apoint of initiation for synthesis of a complementary polynucleotide.Such synthesis occurs when the polynucleotide primer is placed underconditions in which synthesis is induced, e.g., in the presence ofnucleotides, a complementary polynucleotide template, and an agent forpolymerization such as DNA polymerase. A primer is typicallysingle-stranded, but may be double-stranded. Primers are typicallydeoxyribonucleic acids, but a wide variety of synthetic and naturallyoccurring primers are useful for many applications. A primer iscomplementary to the template to which it is designed to hybridize toserve as a site for the initiation of synthesis, but need not reflectthe exact sequence of the template. In such a case, specifichybridization of the primer to the template depends on the stringency ofthe hybridization conditions. Primers can be labeled with a detectablelabel, e.g., chromogenic, radioactive, or fluorescent moieties and usedas detectable moieties. Examples of fluorescent moieties include, butare not limited to, rare earth chelates (europium chelates), Texas Red,rhodamine, fluorescein, dansyl, phycocrytherin, phycocyanin, spectrumorange, spectrum green, and/or derivatives of any one or more of theabove. Other detectable moieties include digoxigenin and biotin.

As used herein a “probe” is defined as a nucleic acid capable of bindingto a target nucleic acid of complementary sequence through one or moretypes of chemical bonds, usually through complementary base pairing,usually through hydrogen bond formation. As used herein, a probe mayinclude natural (i.e. A, G, U, C, or T) or modified bases(7-deazaguanosine, inosine, etc.). In addition, a linkage other than aphosphodiester bond may join the bases in probes, so long as it does notinterfere with hybridization. Thus, probes may be peptide nucleic acidsin which the constituent bases are joined by peptide bonds rather thanphosphodiester linkages. The term “match,” “perfect match,” “perfectmatch probe” or “perfect match control” refers to a nucleic acid thathas a sequence that is perfectly complementary to a particular targetsequence. The nucleic acid is typically perfectly complementary to aportion (subsequence) of the target sequence. A perfect match (PM) probecan be a “test probe”, a “normalization control” probe, an expressionlevel control probe and the like. A perfect match control or perfectmatch is, however, distinguished from a “mismatch” or “mismatch probe.”

A “restriction site” is a portion of a double-stranded nucleic acidwhich is recognized by a restriction endonuclease. A portion of adouble-stranded nucleic acid is “recognized” by a restrictionendonuclease if the endonuclease is capable of cleaving both strands ofthe nucleic acid at a specific location in the portion when the nucleicacid and the endonuclease are contacted. Restriction endonucleases,their cognate recognition sites and cleavage sites are well known in theart. See, for instance, Roberts et al., 2005, Nucleic Acids Research33:D230-D232.

The term “target” as used herein refers to a molecule that has anaffinity for a given probe. Targets may be naturally-occurring orman-made molecules. Also, they can be employed in their unaltered stateor as aggregates with other species. Targets may be attached, covalentlyor noncovalently, to a binding member, either directly or via a specificbinding substance. Examples of targets which can be employed by thisinvention include, but are not restricted to, oligonucleotides andnucleic acids.

“Variant” as the term is used herein, is a nucleic acid sequence or apeptide sequence that differs in sequence from a reference nucleic acidsequence or peptide sequence respectively, but retains essentialproperties of the reference molecule. Changes in the sequence of anucleic acid variant may not alter the amino acid sequence of a peptideencoded by the reference nucleic acid, or may result in amino acidsubstitutions, additions, deletions, fusions and truncations. A variantof a nucleic acid or peptide can be a naturally occurring such as anallelic variant, or can be a variant that is not known to occurnaturally. Non-naturally occurring variants of nucleic acids andpeptides may be made by mutagenesis techniques or by direct synthesis.

Ranges: throughout this disclosure, various aspects of the invention canbe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. Thisapplies regardless of the breadth of the range.

Description

The invention provides compositions and methods for ligating singlestranded nucleic acids wherein the ligation is based on hybridization ofan acceptor molecule with a donor molecule that is fast, efficient, andhas a low-sequence bias. In one embodiment of the invention, thestructure of the donor molecule comprises a stem-loop intramolecularnucleotide base pairing (i.e., hairpin). Therefore, the donor moleculeof the invention is sometimes referred herein as the hairpin donormolecule.

In one embodiment, the acceptor molecule comprises a hydroxyl group atits 3′-terminus and the donor molecule comprises a phosphate at its5′-end. In this manner, the 5′-end of the donor molecule ligates withthe 3′-terminal nucleotide of the acceptor molecule to yield the desiredligation product.

The present invention makes use of a hybridization-based strategywhereby a donor hairpin oligonucleotide is used to hybridize with anacceptor molecule. In one embodiment, the acceptor molecule can be ofany sequence whereas the donor molecule is designed to form a hairpinstructure that includes a 3′-overhang region such that the overhang onthe hairpin oligonucleotide is able to hybridize to nucleotides presentin the 3′ end of the acceptor molecule. Preferably, the hairpin donormolecule having a 3′-overhang region such that the nucleotide(s) foundin the 3′-overhang region of the hairpin oligonucleotide arecomplementary to the nucleotides found in the 3′ end of the acceptormolecule thereby resulting in structure ready for closure by ligation byeither enzymatic or chemical means.

Compositions

In one embodiment, the invention is a nucleic acid hairpin structureuseful for ligating at least two single stranded nucleotides together.In another embodiment, the invention is a nucleic acid structure that isthe result of ligating at least two single stranded nucleotidestogether. The ligation of two single stranded nucleotides involvescombining a first single stranded nucleotide (e.g., acceptor molecule)with a second single stranded nucleotide (e.g., donor molecule), whereinthe second single stranded nucleotide comprises a double stranded regionand a single stranded region. The single stranded region found in thesecond single stranded nucleotide molecule (e.g., donor molecule) is atleast partially complementary to the first single stranded nucleotide(e.g., acceptor molecule). When the acceptor molecule is hybridized tothe donor molecule, the result is a hybrid molecule comprising theacceptor and donor molecule. The hybridized hybrid molecule can beligated as and be subject to further manipulations.

Accordingly, the invention provides a ligation approach that is based onhybridization of two single stranded nucleotides, the first singlestranded nucleotide is referred to as an acceptor molecule and thesecond single stranded nucleotide is referred to as a donor molecule. Insome instances, the acceptor molecule can be of any sequence and thedonor oligonucleotide can be of any sequence and comprises a hairpinstructure.

In one embodiment, the invention includes compositions and methods forligating single stranded nucleic acids wherein the ligation is based onhybridization of an acceptor molecule with a donor molecule that isfast, efficient, and has a low-sequence bias. In one embodiment, thestructure of the donor molecule comprises a stem-loop intramolecularnucleotide base pairing (i.e., hairpin) and a 3′-overhang region suchthat the overhang is able to hybridize to nucleotides present in the 3′end of any acceptor molecule.

Acceptor

The acceptor molecule as well as the donor molecule of the inventioncomprises nucleic acids from any source. A nucleic acid in the contextof the present invention includes but is not limited to deoxyribonucleicacid (DNA), ribonucleic acid (RNA) and peptide nucleic acid (PNA). DNAand RNA are naturally occurring in organisms, however, they may alsoexist outside living organisms or may be added to organisms. The nucleicacid may be of any origin, e.g., viral, bacterial, archae-bacterial,fungal, ribosomal, eukaryotic or prokaryotic. It may be nucleic acidfrom any biological sample and any organism, tissue, cell orsub-cellular compartment. It may be nucleic acid from any organism. Thenucleic acid may be pre-treated before quantification, e.g., byisolation, purification or modification. Also artificial or syntheticnucleic acid may be used. The length of the nucleic acids may vary. Thenucleic acids may be modified, e.g. may comprise one or more modifiednucleobases or modified sugar moieties (e.g., comprising methoxygroups). The backbone of the nucleic acid may comprise one or morepeptide bonds as in peptide nucleic acid (PNA). The nucleic acid maycomprise a base analog such as non-purine or non-pyrimidine analog ornucleotide analog. It may also comprise additional attachments such asproteins, peptides and/or or amino acids.

Donor

In one embodiment, the donor molecule of the invention comprises adouble stranded region and a single stranded region. In one embodiment,the single stranded region is found at the 3′ end of the donor molecule.In one embodiment, the single stranded region is at least partiallycomplementary to a sequence found on an acceptor molecule of theinvention. This complementary sequence found in the donor moleculeallows for the hybridization between the acceptor and donor molecules ofthe invention.

In one embodiment, the donor molecule is a single strandedoligonucleotide that forms an intramolecular stem structure, i.e., ahairpin structure. As used elsewhere herein, a stem structureencompasses a stem-loop structure. Preferably, the intramolecular stemstructure produces a 3′ overhang.

In one embodiment, the donor molecule of the invention comprises: (a) a5′phosphate; (b) a stem or a stem-loop structure; and (c) a 3′ overhang.

In one embodiment, the donor molecule of the invention is a chimericmolecule comprising nucleic acid that has a DNA 5′-end and an RNA 3′-end(or synthetic 3′-end). In another embodiment, the donor moleculecomprises nucleic acid that has an RNA 5′-end and a DNA 3′-end.

3′ Overhang

In one embodiment, the 3′-overhang region of the donor moleculecomprises nucleotides that hybridize to nucleotides found in the 3′ endof the acceptor molecule such that the hybridization between theacceptor molecule and the donor molecule forms a structure referredelsewhere herein as a hybrid molecule of the invention that can beligated by either enzymatic or chemical means.

In one embodiment, the 3′-overhang region comprises at least 1nucleotide, at least 2 nucleotides, at least 3 nucleotides, at least 4nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, atleast 20 nucleotides, at least 25 nucleotides, at least 30 nucleotides,at least 35 nucleotides, or at least 40 nucleotides that arecomplementary to sequences found in the acceptor molecule when theacceptor and donor molecules are hybridized to one another. In thismanner, the 3′-overhang region of the donor molecule is considered asthe region of the donor molecule that binds to the 3′ region of theacceptor molecule.

In various embodiments, the 3′-overhang region comprises at least 1nucleotide, preferably at least 2 nucleotides, preferably at least 3nucleotides, preferably at least 4 nucleotides, and preferably at least5 nucleotides that are mismatched with nucleotides found in the acceptormolecule when the acceptor and donor molecules are hybridized to oneanother.

In one embodiment, the hybridization between the acceptor molecule andthe donor molecule forms a structure that comprises a “nick” wherein thenick can be ligated by either enzymatic or chemical means. A nick in astrand is a break in the phosphodiester bond between two nucleotides inthe backbone in one of the strands of a duplex between a sense and anantisense strand.

In another embodiment, the hybridization between the acceptor moleculeand the donor molecule forms a structure that comprises a “gap” whereinthe gap can be ligated by either enzymatic or chemical means. A gap in astrand is a break between two nucleotides in the single strand.

In one embodiment, the hybridization between the acceptor molecule andthe donor molecule forms a structure that is stable at temperatures thatis as high as 35° C., as high as 40° C., as high as 45° C., as high as50° C., as high as 55° C., as high as 60° C., as high as 65° C., as highas 70° C., as high as 75°, as high as 80° C., as high as 85° C., ormore.

Stem

A donor molecule that is useful in the methods of the inventioncomprises a single-stranded oligonucleotide having a double-strandedportion formed of two self-complementary segments, optionally having aloop at one end, and a short overhanging single strand at the other.Thus, for purposes of the present invention, a hairpin may be defined asa double-helical region formed by nucleotide base-pairing betweenadjacent, inverted, at least partially complementary sequences in asingle-stranded nucleic acid, preferably within the same single strandednucleic acid.

The donor molecule is designed in a manner that the stem structuremaintains its structure prior to and under conditions suitable forhybridization between the donor and acceptor molecules. In this manner,the nick or gap formed through the hybridization between the donor andacceptor molecules can be fixed by way of ligation. In some instances,the donor molecule is designed to also have the stem structure beretained under conditions where the nick or gap is ligated by eitherenzymatic or chemical means. In this situation, a hybrid molecule iscreated by the ligation between the acceptor and donor molecule whereinthe hybrid molecule comprises a larger and more stable stem structurethan was present in the donor alone.

In one embodiment, the intramolecular stem structure preferablymaintains the stem structure under conditions suitable for hybridizationbetween the donor and acceptor molecule. For example, the stem structureis designed to maintain its structure under conditions where theacceptor and donor molecule hybridize.

In some instances, the donor molecule is designed that in someconditions, the intramolecular stem structure has reduced stabilitywhere the stem structure is unfolded. In this manner, the stem structurecan be designed so that the stem structure can be relieved of itsintramolecular base pairing and resemble more of a linear molecule. Inone embodiment, the donor molecule is designed where the relief of theintramolecular stem structure is thermodynamically favored over theintramolecular stem structure. For example, following the generation ofa hybrid molecule of the invention that comprises a stem structure, itis often desirable to amplify or sequence at least a portion of thesequence present in the acceptor molecule portion of the hybrid moleculeof the invention. This can be accomplished by thermodynamicallyrelieving the intramolecular stem structure present in the hybridmolecule by raising the temperature or adding a chemical denaturant.Once the intramolecular stem structure is relieved, a probe or primercan be used to sequence or amplify at least a portion of the sequencepresent in the acceptor molecule.

As discussed elsewhere herein, the stem is designed to form a stablestructure during hybridization and ligation between the acceptor anddonor molecule, yet flexible enough that the stem can be relieved underconditions for amplification or sequencing.

In accordance with the present invention, there are providedpredetermined stem oligonucleotide sequences containing stretches ofcomplementary sequences that form the stem structure. In one embodiment,the stem can comprise at least 3 nucleotide pairs, at least 4 nucleotidepairs, at least 5 nucleotide pairs, at least 6 nucleotide pairs, atleast 7 nucleotide pairs, at least 8 nucleotide pairs, at least 9nucleotide pairs, at least 10 nucleotide pairs, at least 11 nucleotidepairs, at least 12 nucleotide pairs, at least 13 nucleotide pairs, atleast 14 nucleotide pairs, at least 15 nucleotide pairs, at least 20nucleotide pairs, at least 25 nucleotide pairs, at least 30 nucleotidepairs, at least 35 nucleotide pairs, at least 40 nucleotide pairs, atleast 45 nucleotide pairs, at least 50 nucleotide pairs, at least 55nucleotide pairs, at least 60 nucleotide pairs, at least 65 nucleotidepairs, at least 70 nucleotide pairs, at least 75 nucleotide pairs, suchthat these complementary stretches anneal to provide a donor stemoligonucleotide.

In one embodiment, the stem region comprises at least 1 mismatched pair,at least 2 mismatched pairs, at least 3 mismatched pairs, at least 4mismatched pairs, at least 5 mismatched pairs, at least 5 mismatchedpairs, at least 6 mismatched pairs, at least 7 mismatched pairs, atleast 8 mismatched pairs, at least 9 mismatched pairs, at least 10mismatched pairs, at least 11 mismatched pairs, at least 12 mismatchedpairs, at least 13 mismatched pairs, at least 14 mismatched pairs, atleast 15 mismatched pairs, at least 20 mismatched pairs, at least 25mismatched pairs, at least 30 mismatched pairs, at least 35 mismatchedpairs, at least 40 mismatched pairs, at least 45 mismatched pairs, or atleast 50 mismatched pairs.

In one embodiment, it is desirable to have a sufficient amount ofmismatch pairs so that the structure of the stem is unstable at a hightemperature of at least 60° C., at least 65° C., at least 70° C., atleast 75° C., at least 80° C., at least 85° C., at least 90° C., atleast 95° C., at least 96°, at least 97° C., at least 98° C., or atleast 99° C.

In some instances, the donor molecule of the invention comprises astem-loop structure. The loop can comprise any number of nucleotides. Inone embodiment, the loop structure comprises at least 1 nucleotide, atleast 2 nucleotides, at least 3 nucleotides, at least 4 nucleotides, atleast 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, atleast 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, atleast 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides,at least 14 nucleotides, at least 15 nucleotides, at least 20nucleotides, at least 25 nucleotides, at least 30 nucleotides, at least35 nucleotides, or at least 40 nucleotides. Preferably, the loopcomprises about 2-30 nucleotides.

Engineered Features

In one embodiment, the donor molecule of the invention is designed tocomprise a primer binding site. The primer binding site can be designedto be in any, or more than one, region of the donor molecule. In someinstances, it is useful that at least part of the primer binding site bein the loop. This is because base pairs between the primer and loop donot have to compete with base pairs within the stem.

In general, the sequence of the primer binding site can be designed suchthat the sequence thereof is more complementary to a correspondingprimer compared to to any other portion of the acceptor, donor, orhybrid molecules of the invention. The primer binding site can be anylength that supports specific and stable hybridization between theprimer binding site and a primer. For this purpose, a length of about 10to about 35 nucleotides is preferred, with a primer binding site ofabout 16 to about 20 nucleotides long being most preferred.

As discussed elsewhere herein, the stem is designed to form a stablestructure during hybridization and ligation between the acceptor anddonor molecule, yet flexible enough that the stem can be relieved orotherwise unfold under conditions for amplification or sequencing.

Therefore, in some instances, it is desirable to design the donormolecule to have the stem structure be unfolded under suitableconditions for amplification (e.g., suitable denaturing temperatures).For example, following the formation of a ligated hybrid moleculecomprising a donor and acceptor molecule of the invention, it isdesirable to remove the stem structure and thereby create a more linearstructure so that a primer can bind to the corresponding primer bindingsite. For this purpose, it is preferred that the intramolecular stemstructure or stem loop structure be less stable in conditions where itis desirable for a primer to bind to its corresponding primer bindingsite (or, put another way, the hybrid between a primer and a primerbinding site should be more stable than the reformation of theintramolecular stem structure or stem loop structure). For example, whenconditions that promote the hybrid molecule of the invention comprisinga stem structure to lose its intramolecular base pairing and favor amore unfolded structure, it is desirable to have the hybridizationbetween the primer and the primer binding site be more stable than thehybridization between the intramolecular bases within the stem structureof the hybrid molecule. In this way, the primer can bind to the primerbinding site before the stem structure can be reformed in the hybridmolecule.

In another embodiment, once the intramolecular stem structure isrelieved or otherwise is unfolded to resemble an unfolded structure, aprobe or primer can be used to sequence or amplify at least a portion ofthe sequence present in the acceptor portion of the hybrid molecule ofthe invention.

In another, the donor molecule of the invention is designed to comprisea restriction site. The restriction site can be designed to be in any,or more than one, region of the donor molecule or the hybrid productresulting from ligation. Treatment with the corresponding restrictionenzyme can result in the cleavage of the molecule at those residuescorresponding to the restriction enzyme site. The resulting productfollowing restriction enzyme cut can then be manipulated in downstreamreactions, such as for example in cloning, sequencing, or otherwiseinserting the product into a desired plasmid.

In another, the donor molecule of the invention is designed to comprisea label, for example a tag sequence. The tag sequence can be designed tobe in any, or more than one, region of the donor molecule. A tagsequence that is present in the donor molecule is designed so as not tosubstantially impair or interfere with the ability of the donor moleculeto hybridize with the acceptor molecule. Moreover, the tag sequence willbe of sufficient length and composition such that once the tag sequencehas been incorporated into the donor molecule or hybrid molecule of theinvention (e.g., molecule comprising both the acceptor and donormolecule), a tag-specific priming oligonucleotide complementary to thetag can then be used to participate in subsequent manipulation. Skilledartisans will recognize that the design of tag sequences and taggedoligonucleotides for use in the present invention can follow any of anumber of suitable strategies, while still achieving the objectives andadvantages described herein.

In some instances, the tag sequence includes at least one detectablelabel. The label may be any suitable labeling substance, including butnot limited to a radioisotope, an enzyme, an enzyme cofactor, an enzymesubstrate, a dye, a hapten, a chemiluminescent molecule, a fluorescentmolecule, a phosphorescent molecule, an electrochemiluminescentmolecule, a chromophore, a base sequence region that is unable to stablyhybridize to the target nucleic acid under the stated conditions, andmixtures of these.

In other instances, the ligation could be used for sequencing singlemolecules of DNA. If the donor molecule is covalently attached to asolid support, then the acceptor molecule of unknown sequence couldanneal to its templating region and then DNA ligase could be used toseal the nick between the donor and acceptor. The acceptor could then besequenced by next generation sequencing methods.

In yet other instances, the ligation could be used for librarypreparation procedures or yet to be identified molecular biologyprocedures that benefit from a fast, efficient, and low-sequence bias.An example of a library preparation method where the present inventionis applicable can be found in Meyer et al. (2012 Science 338(6104):222-6)).

Methods

This invention relates to ligating single stranded nucleic acids. In oneembodiment, the method comprises: a) contacting a single strandedacceptor nucleic acid molecule with a donor nucleic acid moleculewherein the donor nucleic acid molecule comprises one or more nucleicacids having a double stranded region and a single stranded 3′ terminalregion; b) hybridizing the single stranded 3′ terminal region of thedonor nucleic acid molecule to the acceptor molecule thereby forming anacceptor-donor hybrid molecule comprising a nick or gap between theacceptor nucleic acid and donor nucleic acid molecule; c) and ligatingone 5′ end of the donor nucleic acid molecule to the 3′ end of theacceptor nucleic acid molecule.

In one embodiment, the hybridization between the acceptor molecule andthe donor molecule forms a structure that comprises a nick or gapwherein the nick or gap can be filled and/or ligated by either enzymaticor chemical means.

“Ligation” refers to the joining of a 5′-phosphorylated end of onenucleic acid molecule to a 3′-hydroxyl end of the same or anothernucleic acid molecule by an enzyme called a “ligase.” Alternatively, insome embodiments of the invention, ligation is effected by a type Itopoisomerase moiety attached to one end of a nucleic acid (see U.S.Pat. No. 5,766,891, incorporated herein by reference). The terms“ligating,” “ligation,” and “ligase” are often used in a general senseherein and are meant to comprise any suitable method and composition forjoining a 5′-end of one nucleic acid to a 3′-end of the same or anothernucleic acid.

In addition, ligation can be mediated by chemical agents. Chemicalligation agents include, without limitation, activating, condensing, andreducing agents, such as carbodiimide, cyanogen bromide (BrCN),N-cyanoimidazole, imidazole, 1-methylimidazole/carbodiimide/cystamine,dithiothreitol (DTT) and ultraviolet light. Autoligation, i.e.,spontaneous ligation in the absence of a ligating agent, is also withinthe scope of the teachings herein. Detailed protocols for chemicalligation methods and descriptions of appropriate reactive groups can befound in, among other places, Xu et al., Nucleic Acid Res., 27:875-81(1999); Gryaznov and Letsinger, Nucleic Acid Res. 21:1403-08 (1993);Gryaznov et al., Nucleic Acid Res. 22:2366-69 (1994); Kanaya andYanagawa, Biochemistry 25:7423-30 (1986); Luebke and Dervan, NucleicAcids Res. 20:3005-09 (1992); Sievers and von Kiedrowski, Nature369:221-24 (1994); Liu and Taylor, Nucleic Acids Res. 26:3300-04 (1999);Wang and Kool, Nucleic Acids Res. 22:2326-33 (1994); Purmal et al.,Nucleic Acids Res. 20:3713-19 (1992); Ashley and Kushlan, Biochemistry30:2927-33 (1991); Chu and Orgel, Nucleic Acids Res. 16:3671-91 (1988);Sokolova et al., FEBS Letters 232:153-55 (1988); Naylor and Gilham,Biochemistry 5:2722-28 (1966); and U.S. Pat. No. 5,476,930.

In general, if a nucleic acid to be ligated comprises RNA, a ligase suchas, but not limited to, T4 RNA ligase, a ribozyme or deoxyribozymeligase, Tsc RNA Ligase (Prokaria Ltd., Reykjavik, Iceland), or anotherligase can be used for non-homologous joining of the ends. T4 DNA ligasecan be used to ligate DNA molecules, and can also be used to ligate RNAmolecules when a 5′-phosphoryl end is adjacent to a 3′-hydroxyl endannealed to a complementary sequence (e.g., see U.S. Pat. No. 5,807,674of Tyagi).

If the nucleic acids to be joined comprise DNA and the 5′-phosphorylatedand the 3′-hydroxyl ends are ligated when the ends are annealed to acomplementary DNA so that the ends are adjacent (such as, when a“ligation splint” is used), then enzymes such as, but not limited to, T4DNA ligase, Ampligase™. DNA Ligase (Epicentre Technologies, Madison,Wis. USA), Tth DNA ligase, Tfl DNA ligase, or Tsc DNA Ligase (ProkariaLtd., Reykjavik, Iceland) can be used. However, the invention is notlimited to the use of a particular ligase and any suitable ligase can beused. Still further, Faruqui discloses in U.S. Pat. No. 6,368,801 thatT4 RNA ligase can efficiently ligate DNA ends of nucleic acids that areadjacent to each other when hybridized to an RNA strand. Thus, T4 RNAligase is a suitable ligase of the invention in embodiments in which DNAends are ligated on a ligation splint oligonucleotide comprising RNA ormodified RNA, such as, but not limited to modified RNA that contains2′-F-dCTP and 2′-F-dUTP made using the DuraScribe™ T7 Transcription Kit(Epicentre Technologies, Madison, Wis. USA) or the N4 mini-vRNAP Y678Fmutant enzyme described herein. With respect to ligation on a homologousligation template, especially ligation using a “ligation splint” or a“ligation splint oligonucleotide” (as discussed elsewhere herein), aregion, portion, or sequence that is “adjacent” to another sequencedirectly abuts that region, portion, or sequence.

In some embodiments, a gap of at least one nucleotide is present in theunligated hybrid molecule of the invention that comprises a donormolecule and an acceptor molecule. In some embodiments, the gap isfilled in by a polymerase, and the resulting product ligated. Severalmodifying enzymes are utilized for the nick repair step, including butnot limited to polymerases, ligases, and kinases. DNA polymerases thatcan be used in the methods of the invention include, for example, E.coli DNA polymerase I, Thermoanaerobacter thermohydrosulfuricuspolymerase I, and bacteriophage phi 29. In a preferred embodiment, theligase is T4 DNA ligase and the kinase is T4 polynucleotide kinase.

In one embodiment, ligation of the donor and acceptor molecule involvescontacting the hybridized molecules with a ligase under conditions thatallow for ligation between any two terminal regions of the moleculeswhose 3′ and 5′ ends after hybridization are positioned in a way thatligation may occur.

Any DNA ligase is suitable for use in the ligation step. Preferredligases are those that preferentially form phosphodiester bonds at nicksin double-stranded DNA. That is, ligases that fail to ligate the freeends of free single-stranded DNA at a significant rate are preferred. Insome instances, thermostable ligases can be used. In other instances,thermosensitive ligases are preferred because the ligase can be heatinactivated. Many suitable ligases are known, such as T4 DNA ligase(Davis et al., Advanced Bacterial Genetics—A Manual for GeneticEngineering (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.,1980)), E. coli DNA ligase (Panasnko et al., J. Biol. Chem.253:4590-4592 (1978)), AMPLIGASE™ (Kalin et al., Mutat. Res., 283(2):119-123 (1992); Winn-Deen et al., Mol Cell Probes (England) 7(3):179-186(1993)), Taq DNA ligase (Barany, Proc. Natl. Acad. Sci. USA 88:189-193(1991), Thermus thermophilus DNA ligase (Abbott Laboratories), Thermusscotoductus DNA ligase and Rhodothernius marinus DNA ligase(Thorbjarnardottir et al., Gene 151:177-180 (1995)). T4 DNA ligase ispreferred for ligations involving RNA target sequences due to itsability to ligate DNA ends involved in DNA:RNA hybrids (Hsuih et al.,Quantitative detection of HCV RNA using novel ligation-dependentpolymerase chain reaction, American Association for the Study of LiverDiseases (Chicago, Ill., Nov. 3-7, 1995)).

Amplification

The ligation product that comprises the donor and acceptor molecule canbe isolated or amplified using a primer that corresponds to a primerbinding site present in the ligated product (i.e., primer binding sitepresent in the donor molecule or the resulting hybrid product).

In particular embodiments of the invention the quantifying stepscomprise a method selected from the group consisting of gelelectrophoresis, capillary electrophoresis, labelling reactions withsubsequent detection measures and quantitative real-time PCR orisothermal target amplification. Preferably, the quantification stepscomprise quantitative real-time PCR or quantitative real-time isothermalamplification. More preferably, quantification comprises quantitativereal-time PCR.

The ligation product or otherwise the template nucleic acid may beamplified, while attached or unattached to beads, by any suitable methodof amplification including transcription-based amplification systems(Kwoh D. et al., Proc. Natl. Acad. Sci. (U.S.A.) 86:1173 (1989);Gingeras T. R. et al., WO 88/10315; Davey, C. et al., EP Publication No.329,822; Miller, H. I. et al., WO 89/06700), “RACE” (Frohman, M. A., In:PCR Protocols: A Guide to Methods and Applications, Academic Press, NY(1990)) and one-sided PCR (Ohara, O. et al., Proc. Natl. Acad. Sci.(U.S.A.) 86.5673-5677 (1989)). Still other methods such asdi-oligonucleotide amplification, isothermal amplification (Walker, G.T. et al., Proc. Natl. Acad. Sci. (U.S.A.) 89:392-396 (1992)), NucleicAcid Sequence Based Amplification (NASBA; see, e.g., Deiman B et al.,2002, Mol. Biotechnol. 20(2):163-79), whole-genome amplification (see,e.g., Hawkins T L et al., 2002, Curr Opin Biotechnol. 13(1):65-7),strand-displacement amplification (see, e.g., Andras S C, 2001, Mol.Biotechnol. 19(1):29-44), rolling circle amplification (reviewed in U.S.Pat. No. 5,714,320), and other well-known techniques may be used inaccordance with the present invention. In certain aspects, a nucleicacid template is amplified after encapsulation with a bead in amicroreactor. Alternatively, a nucleic acid template is amplified afterdistribution onto a multiwell surface, e.g., a PicoTiter plate.

In a preferred embodiment, DNA amplification is performed by PCR. PCRaccording to the present invention may be performed by contacting thetarget nucleic acid with a PCR solution comprising all the necessaryreagents for PCR. Then, PCR may be accomplished by exposing the mixtureto any suitable thermocycling regimen known in the art. In a preferredembodiment, 30 to 50 cycles, preferably about 40 cycles, ofamplification are performed. It is desirable, but not necessary, thatfollowing the amplification procedure there be one or more hybridizationand extension cycles following the cycles of amplification. In apreferred embodiment, 10 to 30 cycles, preferably about 25 cycles, ofhybridization and extension are performed (e.g., as described in theexamples). Routinely, the template DNA is amplified until typically atleast 10,000 to 50,000,000 copies are immobilized on each bead. It isrecognized that for nucleic acid detection applications, fewer copies oftemplate are required. For nucleic acid sequencing applications weprefer that at least two million to fifty million copies, preferablyabout ten million to thirty million copies of the template DNA areimmobilized on each bead. The skilled artisan will recognize that thesize of bead (and capture site thereon) determines how many captiveprimers can be bound (and thus how many amplified templates may becaptured onto each bead).

In particular embodiments of the invention the polymerase used forquantitative real-time PCR is a polymerase from a thermophile organismor a thermostable polymerase or is selected from the group consisting ofThermus thermophilus (Tth) DNA polymerase, Thermus acquaticus (Taq) DNApolymerase, Thermotoga maritima (Tma) DNA polymerase, Thermococcuslitoralis (Tli) DNA polymerase, Pyrococcus furiosus (Pfu) DNApolymerase, Pyrococcus woesei (Pwo) DNA polymerase, Pyrococcuskodakaraensis KOD DNA polymerase, Thermus filiformis (Tfl) DNApolymerase, Sulfolobus solfataricus Dpo4 DNA polymerase, Thermuspacificus (Tpac) DNA polymerase, Thermus eggertssonii (Teg) DNApolymerase, Thermus brockianus (Tbr) and Thermus flavus (Tfl) DNApolymerase.

In preferred embodiments of the invention the primer or probe islabelled with one or more fluorescent dye(s) and/or quencher(s) andwherein the quantifying steps comprise detecting fluorescence signals inthe sample.

Particularly, the fluorescently labelled primers or probes are labelledwith a dye selected from the group consisting of FAM, VIC, NED,Fluorescein, FITC, IRD-700/800, CY3, CY5, CY3.5, CY5.5, HEX, TET, TAMRA,JOE, ROX, BODIPY TMR, Oregon Green, Rhodamine Green, Rhodamine Red,Texas Red, Yakima Yellow, Alexa Fluor and PET or analogous dyes withsimilar excitation and emission properties.

In one embodiment, the primer or probe is a LightCycler probe (Roche) orthe hydrolysis probe is a TaqMan probe (Roche). In other embodiments theprimer or probe includes but is not limited to molecular beacon,Scorpion primer, Sunrise primer, LUX primer and Amplifluor primer.

Applications

The ssDNA ligation composition and method of the present invention maybe used in a wide variety of protocols and technologies. For example, incertain embodiments, ssDNA ligation is used in the fields of molecularbiology, genomics, transcriptomics, epigenetics, nucleic acidsequencing, and the like. That is, ssDNA ligation may be used in anytechnology that may require or benefit from the ligation of ssDNA.Exemplary technologies, include, but are not limited toLigation-Mediated PCR (LMPCR); cDNA library construction; DNA epigenome(such as m5C) and RNA methylome (such as m6A) assays, high-throughputnext generation sequencing technologies including but not limited toIllumina, SOLiD, and Ion Torrent sequencing; and single nucleic acidmolecule real time sequencing (SMRT) including, but not limited to,technologies from Pacific Bioscience and Oxford Nanopore Technologiessuch as zero-mode waveguide or nanopore sequencing, respectively.

In one embodiment, the ssDNA ligation composition and method of theinvention is applicable to DMS/SHAPE-LMPCR and Structure-Seq, andDMS-seq. These technologies are described, for example, in Kwok et al.(Kwok et al, 2013, Nature Communications, 4: article number: 297), Dinget al. (Ding et al., 2013, Nature, November 24. doi:10.1038/nature12756), and Rouskin et al. (Rouskin et al., 2013, Nature,doi:10.1038/nature12894), respectively, the contents of which areincorporated by reference herein in their entirety.

In one embodiment, the ssDNA ligation composition and method of theinvention can be used in a DMS/SHAPE-LMPCR method to determine RNAstructure in vivo and in vitro in low-abundance transcripts, for anyorganism or tissue.

In another embodiment, the ssDNA ligation composition and method of theinvention can be used in Structure-Seq, a method that allows forgenome-wide profiling of RNA secondary structure, both in vivo and invitro, for any organism or tissue.

In another embodiment, the ssDNA ligation composition and method of theinvention can be used in DMS-Seq, another method that allows genome-wideprobing of RNA secondary structure, both in vivo and in vitro, in anyorganism or tissue.

Kits

The present invention also relates to a kit for performing any of theabove described methods, wherein the kit comprises one or more of: (a) adonor molecule; (b) a ligase; and, optionally, (c) a primersubstantially complementary to a primer binding site present in thedonor molecule

In one embodiment, the kit additionally comprises a ligase. In anotherembodiment, the kit additionally comprises a polymerase. The kit mayadditionally also comprise a nucleotide mixture and (a) reactionbuffer(s) and/or a set of primers and optionally a probe for theamplification and detection of the ligation product between an acceptorand donor molecule.

In particular embodiments, the kit additionally comprises one or morepre-quantified calibrator nucleic acids, a set of primers for theamplification of said calibrator nucleic acids and a first nucleic acidprobe substantially complementary to a sequence on said pre-quantifiednucleic acid.

In some embodiments, one or more of the components are premixed in thesame reaction container.

EXPERIMENTAL EXAMPLES

The invention is further described in detail by reference to thefollowing experimental examples. These examples are provided forpurposes of illustration only, and are not intended to be limitingunless so specified. Thus, the invention should in no way be construedas being limited to the following examples, but rather, should beconstrued to encompass any and all variations which become evident as aresult of the teaching provided herein.

Without further description, it is believed that one of ordinary skillin the art can, using the preceding description and the followingillustrative examples, make and utilize the compounds of the presentinvention and practice the claimed methods. The following workingexamples therefore, specifically point out the preferred embodiments ofthe present invention, and are not to be construed as limiting in anyway the remainder of the disclosure.

Example 1 : A Hybridization-Based Approach for Quantitative and Low-BiasSingle-Stranded DNA Ligation

Single-stranded (ss) DNA ligation is a crucial step in many biochemicalassays. Efficient ways of carrying out this reaction are lacking,however. As demonstrated herein, existing ssDNA ligation methods sufferfrom slow kinetics, poor yield, and severe nucleotide preference. Toresolve these issues, a hybridization-based strategy is presentedherein, which provides efficient and low-bias ligation of ssDNA. Theligation approach presented herein is based on hybridization of anincoming acceptor DNA oligonucleotide to a hairpin DNA using T4 DNAligase, which is fast, efficient, low-bias, and integrates seamlesslywith downstream protocols. This technique can be applied in protocolsthat require ligation of ssDNA, including Ligation-Mediated PCR (LMPCR),and cDNA library construction. The technique could also be used in avariety of high-throughput, next generation sequencing technologiesincluding, but not limited to, Illumina, SOLiD, and Ion Torrentsequencing as well as the sequencing of single molecules of DNAincluding, but not limited to, technologies from Pacific Bioscience suchas SMRT.

The materials and methods employed in these experiments are nowdescribed.

DNA Oligonucleotides and Purification

All PAGE-purified 24 nucleotide Cy5-labeled acceptor DNA were purchasedfrom Sigma Aldrich. All remaining DNAs were from Integrated DNATechnologies (IDT). All donor oligonucleotides were purified by 10%urea-polyacrylamide gel electrophoresis, and the bands were excisedindividually under UV shadowing, which was brief in order to preventformation of photolesions (Kladwang et al., 2012, Sci. Rep. 2:517). Eachband was crushed and soaked in 10 mM Tris, pH 7.5, 1 mM EDTA, 250 mMNaCl (1×TEN₂₅₀) overnight at room temperature with constant rotaryshaking. The gel mixture was filtered against a 0.25 μm filter,subjected to ethanol precipitation by addition of 3× volume of 100%ethanol, and frozen in dry ice for an hour. The frozen slurry wassubsequently centrifuged at 13,000 rpm for 20 min, and the pellet washedwith cold 70% ethanol to remove residual salts. Residual ethanol wasremoved by speed-vac for 5-10 min, and the pellet was dissolved in waterand quantified with UV-spectroscopy to determine the DNA concentration.DNAs were stored at −20° C. both before and after experiments, and thevials containing the Cy5 and FAM labeled acceptor DNAs were wrapped withaluminum foil to prevent photobleaching, and stored at −20° C. bothbefore and after use.

Ligation Reaction and Data Collection

Circligase I:

The reaction contained 100 pmol of 5′p donor, 1 pmol of Cy5-labelledacceptor, 50 mM MOPS (pH 7.5), 10 mM KCl, 5 mM MgCl₂, 1 mM DTT, 0.05 mMATP, 2.5 mM MnCl₂ and 200 U Circligase I. The reaction was performed at65° C. for 12 h, and then 85° C. for 15 min to deactivate the enzyme. InFIG. 7, 68° C. was used instead, and 20% PEG 8000 was included.

T4 RNA Ligase I:

The reaction contained 100 pmol of 5′p donor, 1 pmol of Cy5-labelledacceptor, 50 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 1 mM ATP, 1 mM DTT, and20 U T4 RNA ligase I. The reaction was performed at 37° C. for 12 h, andthen 65° C. for 15 min to deactivate the enzyme.

T4 DNA Ligase:

The ligation reaction contained 100 pmol of 5′p donor, 1 pmol ofCy5/FAM-labelled acceptor, 50 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 10 mMDTT, 1 mM ATP, and 15 U T4 DNA ligase. Ligation factors such as PEG8000, betaine, temperature, and time were tested as described elsewhereherein (FIGS. 9-15). The reaction was performed at 16° C. for 12 h, andthen 65° C. for 15 min to deactivate the enzyme. Unless otherwiseindicated, a laboratory preparation of T4 DNA ligase was used.

All ligation reactions were quenched by 2× formamide dye with 20 mMEDTA. Samples were heated at 95° C. for 1.5 min, and 3 μA was loaded toan 8.3 M urea 10% polyacrylamide gel. The pre-heated gel (surfacetemperature at ˜50-60° C.) was subjected to electrophoresis at 900-1000V (constant) for 10-15 min, and then was directly scanned using the redlaser (633 nm) of a Typhoon PhosphorImager 9410 and a 670 nm emissionfilter (BP 30 nm). The plate focus was set at 3 mm.

Data Processing and Analysis

The background corrected unligated (U) and ligated (L) bands werequantified using ImageQuant 5.2, and the ligation efficiency wascalculated based on band intensities using the following equation:

$\begin{matrix}{{{Ligation}\mspace{14mu}{efficiency}} = {\frac{L}{U + L}.}} & \left( {{Equation}\mspace{14mu} 1} \right)\end{matrix}$

The plots of fraction ligated versus time were fit to a singleexponential equation in KaleidaGraph 3.5. The equation is as follows:Fraction ligated=A+Be ^(−kobs(t))  (Equation 2),

where k_(obs) is the observed first-order rate constant for ligation forthe non-burst phase, t is time, A is the fraction of ligated product atcompletion, −B is the amplitude of the observable phase, 1−A is theunreactive fraction, and A+B is the burst fraction (Chadalavada et al.,2010, Biochemistry 49:5321-5330).

The results of the experiments are now described.

ssDNA Ligation Using Circligase I and T4 RNA Ligase I

To assay for ligation, two ssDNA oligonucleotides were designed,referred to as “acceptor” and “donor” (FIG. 1A). The acceptoroligonucleotide contains a Cy5 fluorophore at its 5′-terminus and ahydroxyl group at its 3′-terminus, whereas the donor oligonucleotide hasa phosphate at its 5′-end and a C3-spacer group at its 3′-end thatprevents donor self-oligomerization. For convenience of this ligationassay, these acceptor and donor oligonucleotides are relatively small,at 24 and 33 nucleotides, respectively, although any size is possible,as shown later. The Mfold-predicted secondary structures (Zuker, 2003,Nucleic Acids Res. 31:3406-3415) and the sequences of the initialacceptor and donor oligonucleotides are provided in FIG. 1A, and theirimportance for ssDNA ligation is tested below.

First, the efficiency of Circligase I was tested (FIG. 1B). Ligation ofssDNA with each of the four DNA bases at the 3′ end of the acceptor wasassessed after 12 h of incubation under the vendor's recommendedcondition, as discussed elsewhere herein. The yield varied widely, from18-73%, with a strong acceptor 3′ end bias of dT>dA>>dG>>dC (FIG. 1B).Without being bound to any particular theory, it is believed that thistrend matched that of the DNA circularization reaction using CircligaseI. Similar or slightly improved yields and nucleotide bias were observedwhen another donor oligonucleotide with minimal secondary structure wasused (ranging from 32-86%), or when the first 3 nucleotides of the donorsequence were randomized (ranging from 48-88%) (FIG. 6). Theseobservations suggest that the pooled donor approach used elsewhere(Jayaprakash et al., 2011, Nucleic Acids Res. 39(21):e141) or that usinga donor with minimal secondary structure cannot appreciably alleviatethe inherent nucleotide bias. In fact, the acceptor Mfold predictedstructure (FIG. 1A) did not reveal strong secondary structure at its 3′end, and ligation reaction for Circligase I was performed at 65° C.,which likely denatures secondary structure. These findings suggest thatsuch nucleotide bias at the 3′-end of the acceptor is a common featurefor Circligase and not due to secondary structure. It was previouslyreported that Circligase I is a RNA ligase that shows homology to T4 RNAligase I (Blondal et al., 2005, Nucleic Acids Res. 33:135-142), and itis well known that T4 RNA ligase I has strong preference to ligate tocertain end nucleotide over others (Ohtsuka et al, 1977, Eur. J.Biochem. 81: 285-291; Harada et al., 1993, Proc. Natl. Acad. Sci. USA90:1576-1579; McLaughlin et al., 1982, Eur. J. Biochem. 125:639-643;Rieder et al., 2009, Methods Mol. Biol. 540:15-24), consistent withthese observations.

To test the properties of Circligase further, the experiments presentedin FIG. 1B were repeated in the presence of 20% PEG 8000 and at 68° C.(Li et al., 2006, Anal. Biochem. 349:242-246). It was found thatnucleotide bias was still obvious and the bias spanned 25-64% after 12 h(FIG. 7). These results all suggested that the nucleotide bias inCircligase ssDNA ligation cannot be remediated by rational experimentaldesign. This may not pose a problem for certain applications, such as ifquantitative analysis is not required, as in 5′ rapid amplification ofcDNA ends (D. Bertioli, R. Rapley. (2000) “Rapid Amplification of cDNAEnds” The Nucleic Acid Protocols Handbook, pp 613-617, Humana Press.),where only the 5′ most nucleotide of the RNA is of major concern.Notably, without wishing to be bound by any particular theory, ifCircligase I is used for ssDNA ligation with acceptors of unknown 3′end, severe sequence bias will result in which the sequence of the RNAsof interest will not be properly represented in the final reactionproducts. Since Circligase II is the same protein as Circligase I,differing only in adenylation status, it is likely that severe sequencebias will also result if Circligase II is used for ssDNA ligation withacceptors of unknown 3′ end.

Next, T4 RNA ligase-mediated ligation of ssDNA was examined. Here theyield was very poor: the ligated product was only ˜1% under the examinedconditions (FIG. 1C). As such the nucleotide preference cannot beassessed. Consistent with observations presented herein, T4 RNA ligase Iwas previously shown to be less efficient than Circligase I for ssDNAligation (Blondal et al., 2005, Nucleic Acids Res. 33:135-142) andrequired PCR amplification to obtain observable products (Zhang et al.,1996, Nucleic Acids Res. 24:990-991). It is noted that both Circligase-and T4 RNA ligase-mediated ligation of ssDNA occur in atemplate-independent fashion, as depicted in at the bottom of FIG.1B-FIG. 1D.

Initial Tests of ssDNA Ligation Using T4 DNA Ligase

Next, ssDNA ligation using T4 DNA ligase was attempted. To obtain theoptimal condition for ligation, factors that could contribute toligation efficiency were tested. It was found that 20% PEG 8000, 0.5Mbetaine, and 100:1 donor:acceptor ratio resulted in high (>80%) ligationyield of the acceptor at 16° C. and 3 h (FIGS. 9-15). Initially, thenucleotide preference test described above for Circligase I showed thatT4 DNA ligation was efficient only when the acceptor 3′-end was a “G”residue (FIG. 1D). It is well-known that T4 DNA ligase mostly acts on adsDNA junction (Nilsson et al., 1982, Nucleic Acids Res. 10: 1425-1437;Alexander et al., 2003, Nucleic Acids Res. 31:3208-3216; Lehman, 1974,Science 186:790-797), although template-independent T4 DNA ligation ofssDNA has also been reported, albeit at a very low efficiency (Kuhn andFrank-Kamenetskii, 2005, FEBS J 272:5991-6000). Sequence-specificligation (FIG. 1D, top) suggested that intra- and intermolecular DNAsecondary structure could be influencing the ligation. This suggested amodel in which a dsDNA junction was formed between the donor andacceptor for ligation to occur (FIG. 1D, bottom).

To better understand the ligation result, Mfold was used to predict thesecondary structure of the ligated DNA product. This revealedintermolecular base pairing of the acceptor and donor, similar to aprimer-template model. While not wishing to be bound by any particulartheory, a possible mode of reaction was shown in FIG. 2. From thismodel, it is reasoned that the low ligation efficiency with the otherbases at the 3′-end of the acceptor (FIG. 1D, top) might be due to thepresence of a mismatch between the acceptor and donor at the ligationjunction, which is herein referred to as 24′:18. Although the ligationusing T4 DNA ligase requires base pairing at the junction, this reactionis herein regarded as ssDNA ligation because both acceptor and donor aresingle strands.

Hairpin Donor Design and Mutational Studies

To test the hypothesis that acceptor-donor mismatch was impairingligation of non-G terminated acceptors, a series of mutational studieswere performed. First, it was attempted to rescue ligation efficiency ofacceptor 3′-ends T, C, and A by restoring the 24′:18 base pairing (FIG.2; see mutants M1, M2, and M3, respectively). M1 and M3 showed markedimprovements in yield, from just 1% and 2% to 81% and 67%, respectively(FIGS. 2 and 16). M2, however, still had a low yield of 2%. While notwishing to be bound by any particular theory, secondary structureprediction revealed that this was likely due to an alternative structureof the donor, which hindered base pairing of the acceptor. In an effortto limit this alternative structure, the native hairpin structure of thedonor was strengthened by introducing Watson-Crick (WC) base pairing atposition 4:14, far from the site of ligation (FIG. 2) (see M13-M15). Itis shown that these changes were compatible with efficient ligation inthe presence of a 3′-end G acceptor, where they actually increased thekinetics of ligation somewhat (compare 1 min lanes in FIGS. 16 and 20).Importantly, WC base pairing at 4:14 rescued M2, (see M16=M15+M2) with aligation efficiency of 89% (FIGS. 2 and 16). Thus, native donor hairpinstructure contributes to achieving efficient ligation. After confirmingthat all WC base pairs at 24′:18 are efficiently incorporated, the 4homonucleobase mismatches at the 24′:18 base pair were tested (seeM4-M7). None of these mutants gave appreciable ligated product, evenafter 12 h (just 1-3% yield) indicating that the T4 DNA ligation ofssDNA has fidelity of base pair recognition with the 3′-end of theacceptor (FIGS. 2 and 17).

Using the same rationale, the importance of the 5′-end of the donor wastested for ligation efficiency via the terminal base pair 1:17 (FIG. 2).Efficient ligation was observed with all three other WC base pairs at1:17, M8-M10 (FIGS. 2 and 18). Changing the 1:17 base pair to an AA orTT mismatch, however, reduced the ligation yield to 9 and 36%,respectively (FIGS. 2 and 19). While not wishing to be bound by anyparticular theory, the intermediate yield observed for the TT mismatchat the 1:17 base pair is likely due to formation of a two hydrogen bondwobble base pair owing to intramolecular folding of the donor. Such anintramolecular wobble TT mismatch formation has been observed previouslyby NMR spectroscopy (He et al., 2011, FEBS Lett. 585:3953-3958).Overall, the T4 DNA ligation of ssDNA has fidelity of base pairing forboth base pairs flanking the ligation junction.

Lastly, the importance of the 3′-end and the loop of the donor wastested. The ligation yield of a 3′-end deletion mutant, M17, was 81%after 12 h, although the kinetics was somewhat slower (FIGS. 20 and 21).While not wishing to be bound by any particular theory, one possiblereason for a slower rate is that the 3′-overhang present in the fulllength donor, but absent in the mutant, forms a mini-hairpin (FIG. 1)that may reduce alternative structure. A loop mutant, M18, that changedthe apical T to the larger non-WC base paired hexyloop of CTAGTC wasmade and had a slightly increased yield of 86%, which, while not wishingto be bound by any particular theory, could be due to formation of the8:10 CG base pair, previously precluded by the smaller loop (FIGS. 2 and21) (Shu and Bevilacqua, 1999, Biochemistry 38:15369-15379). There aretwo potential advantages to having a larger hairpin loop in the donor:i) it can help avoid donor-donor dimerization, which could hinderligation, and; ii) it can provide part of a primer binding site and thusincrease the melting temperature of the primer used in PCR fordownstream applications.

Optimized ssDNA Ligation Using T4 DNA Ligase

For application to a LMPCR type experiment, the 3′ end of the acceptoris likely to be truncated and thus have different sequences from thefull-length cDNA generated from reverse transcription. Therefore, ageneral donor oligonucleotide (40 mer) was designed by introducing arandom hexamer region that can hybridize with different incomingacceptors. It was tested whether this donor could ligate efficiently andin an unbiased manner to various acceptors (FIG. 3A). This donor alsocontains the mini-hairpin as a 3′-overhang since this promoted moreefficient kinetics. The rationale for constructing a random hexamerregion to widely target acceptor sequences in single-stranded DNAligation is similar to the idea for random hexamer priming in reversetranscription, wherein use of random hexamers enables targeting ofdiverse RNA sequences. To evaluate this donor, the nucleotide preferenceligation test was performed on four 24 mer acceptors each with adifferent base at its 3′ end. The ligation efficiencies for theindividual acceptors were very similar to one another, with averageyields of 93±1%, 90±1%, 96±1%, 95±1% for A, G, C and T, respectively(FIG. 3B). This outcome contrasts sharply with the data in FIG. 1D,where only one of the four acceptors ligated efficiently. Moreover, thisresult provides much less bias as compared to Circligase I (FIG. 1A,FIG. 6 and FIG. 7).

Next the kinetics of the T4 DNA ligase-mediated ssDNA ligation reactionwas tested using the optimized donor with a hexyloop and a randomhexamer template region. A first-order rate constant of >2 h⁻¹, or >50%reaction in less than 30 min (FIGS. 3C and 22), was found. To evaluatewhether this optimized donor can ligate to other acceptors, 3 additionalacceptors with varying sequence were tested, and it was found that theyield were just as efficient as mentioned above (FIG. 4). Given thequantitative and low-bias nature of the template-mediated approach, asillustrated in FIG. 3, FIG. 4, and FIG. 22, this approach differssignificantly from the Circligase method mentioned above, in notrequiring correction for nucleotide bias with a statistical correctionfactor.

Potential Applications of Optimized Hairpin Donor

To test the general applicability of the ssDNA ligation method describedherein, it was sought to mimic ligation of the longer cDNAs that wouldbe generated during reverse transcription. A 91 nucleotide DNA acceptorwas designed that contains hydroxyl groups at both termini. Thisacceptor was subjected to ssDNA ligation with the 40 mer donor from FIG.3, and product formation was followed by PCR. A 131-bp PCR productformed in a T4 DNA ligase-dependent fashion (FIG. 5). This resultindicated that the hairpin donor can be used for LMPCR. It should benoted that although this approach requires a hairpin stem and a randomtemplating region, the hairpin stem can be varied to other WC base paircombination (see FIG. 2) to avoid any sequence complementary to the geneof interest and mis-priming during PCR reaction. Also, it should benoted that any strong secondary structure at the 3′ end of the DNAacceptor may interfere with the hybridization and thus the ligationefficiency. A locked nucleic acid (LNA) base at the randomizedtemplating region may be designed and introduced to allow more efficienthybridization (Fratczak et al., 2009, Biochemistry 48(3):514-6). Inaddition, low amounts of DMSO (5-10%) often help to improve reactivityof protein enzymes that act on nucleic acids, such as tRNA ligase andpolynucleotide kinase (PNK), at the ends of structured RNAs (Bruce andUhlenbeck, 1978, Nucleic Acids Res. 5:3665-3678; Strauss et al., 1968,Biopolymers 6:793-807).

It is noted that for next generation sequencing, the platform-specificadaptor (donor) has to be ligated in an early step of cDNA libraryconstruction, such as in the SHAPE-Seq application (Lucks et al., 2011,Proc. Natl. Acad. Sci. USA 108:11063-11068). Various sequencingplatforms for next-generation sequencing are available, and the choiceof a specific platform will likely be influenced by sequencing cost,instrument availability, and user application. Therefore, duringdevelopment of the hairpin donor described herein, a 7 nucleotide TypeIIS restriction site (SapI/BspQI) was incorporated, which provides theuser an option to remove the donor after PCR amplification, if needed(FIGS. 3 and 5). On the one hand, the advantage of retaining the donorsequence, including the restriction enzyme site, is that it can providean internal check of the 3′ end of the acceptor DNA, as the sequencingresult should have donor sequence immediately followed by acceptorsequence. On the other hand, if the donor is removed by SapI/BspQIrestriction digest, the digested dsDNA pool can also be subjected toroutine processing as required for the production of next-generationsequencing libraries, including the steps of end-repair, dA tailing andplatform sequence-specific double-stranded adaptor ligation. Thisgeneral approach allows the decision for next generation sequencingplatform to be made at the last step, and allows cross-platformvalidation (Linsen, et al., 2009, Nat. Meth. 6:474-476; Potapova et al.,2011, BMC Biotechnology 11:6) on the same or different cDNA librarysample, as the digested dsDNA pool can be re-used for differentplatforms. This is in contrast to some cDNA library preparation methods,such as SHAPE-Seq, in which a platform-specific adaptor sequences has tobe ligated in an early step and such adaptor sequences usually only workin a single platform. Lastly, the high throughput data obtained willhave greatly reduced sequence bias.

In summary, described herein is a fast, efficient, and low-bias methodfor ligating two ssDNAs. A hairpin donor DNA hybridizes with differentacceptor 3′-end to yield the desired ligation product (FIG. 3B). Thereaction uses the common enzyme T4 DNA ligase and is completed in 2 h.The method provides an alternative approach for ssDNA ligations that canbe applied to LMPCR, and allows platform-free cDNA library construction,including for cross-platform validation. It can also be used as a toolto develop new biochemical and molecular biology methods. Lastly, thisssDNA ligation method can also be used in a sequence-specific mode toallow only certain acceptor 3′-ends to give a ligation product (e.g.FIG. 1D), which provides an approach for selecting specific sequencesfrom a 3′-end pool.

Example 2 : Determination of In Vivo RNA Structure in Low-AbundanceTranscripts

RNA is of central importance in gene regulation, catalysis and theorigin of life (Gesteland, R. F., et al., Cold Spring Harbor LaboratoryPress, 2006.). Numerous classes of RNA perform key biological functionsvia folding into diverse structures. Knowledge of RNA structure in vivotherefore provides important insights regarding the evolution andfunction of biological systems. However, the structures of all but thefew most abundant RNAs have been unknown in vivo.

For decades, chemical and enzymatic probing have been among the mostcommon and powerful assays available to obtain structural information onRNA at nucleotide resolution (Ehresmann, C. et al., Nucleic Acids Res.15:9109-9128, 1987; Stern, S., et al., Science 244:783-790, 1989; Weeks,K. M., Curr. Opin. Struct. Biol. 20:295-304, 2010 and Ding, F., et al.,Nat. Meth. 9:603-608, 2012). This information can dramatically improvesecondary structure prediction (Mathews, D. H. et al., Proc. Natl. Acad.Sci. USA 101:7287-7292, 2004; Low, J. T. et al., Methods 52:150-158,2010 and Cordero, P., et al., Biochemistry 51:7037-7039, 2012).Structures generated provide insights regarding the control of RNAtranscription, processing, stability, translation and ligand-binding.

Among RNA structural probing reagents, dimethyl sulfate (DMS) is highlyversatile and useful for in vivo probing (Zemora, G. et al., RNA Biol.7:634-641, 2010), owing to its ability to penetrate cells and modify RNAin numerous organisms (Moazed, D., et al., Nature 334:362-364, 1988;Senecoff, J. F. et al., Plant Mol. Biol. 18:219-234, 1992; Zaug, A. J.et al., RNA 1:363-374, 1995; Higgs, D. C., et al., Mol. Cell Biol.19:8479-8491, 1999; Wells, S. E., et al., Methods Enzymol. 318:479-493,2000; Iseni, F., et al., RNA 6:270-281, 2000 and Antal, M., et al.,Nucleic Acids Res. 30:912-920, 2002). Recently, in vivo SHAPE reagentswere developed and have been used to probe the highly abundant 5S rRNAin bacteria, yeast, fly and mammalian cells (Spitale, R. C. et al., Nat.Chem. Biol. 9:18-20, 2013). DMS methylates the N1 of adenine and the N3of cytosine on the Watson-Crick base pairing face of unprotected regionssuch as loops, bulges and mismatches (Zaug, A. J. et al., RNA 1:363-374,1995 and Wells, S. E., et al., Methods Enzymol. 318:479-493, 2000),whereas SHAPE reagents acylate the 2′-hydroxyl group on the ribose sugarof unstructured regions of all four nucleotides (Spitale, R. C. et al.,Nat. Chem. Biol. 9:18-20, 2013 and Wilkinson, K. A., et al., Nat.Protoc. 1:1610-1616, 2006). Methylation or acylation chemistry isdetected by reverse transcription (RT) stops one nucleotide before themodified nucleotide (Zaug, A. J. et al., RNA 1:363-374, 1995; Wells, S.E., et al., Methods Enzymol. 318:479-493, 2000; Spitale, R. C. et al.,Nat. Chem. Biol. 9:18-20, 2013; Wilkinson, K. A., et al., Nat. Protoc.1:1610-1616, 2006 and Inoue, T. et al., Proc. Natl. Acad. Sci. USA82:648-652, 1985).

In cellular systems, structures of high-abundance RNAs such as rRNA canbe assessed in vivo by a DMS/SHAPE-RT approach (Senecoff, J. F. et al.,Plant Mol. Biol. 18:219-234, 1992; Zaug, A. J. et al., RNA 1:363-374,1995; Wells, S. E., et al., Methods Enzymol. 318:479-493, 2000 andSpitale, R. C. et al., Nat. Chem. Biol. 9:18-20, 2013). However, thevast majority of RNAs in a typical cell are of low abundance in vivo andcannot be explored by an RT-based approach. As such, very little isknown about the in vivo structures of myriad RNAs, including most mRNAsand non-coding (nc) RNAs, despite their essential roles in proteinsynthesis and other cellular processes. Moreover, the effects ofRNA-binding proteins on in vivo RNA structures are also largelyunexplored.

As described by Kwok et al., (Kwok et al, 2013, Nature Communications,4: article number: 297), the contents of which are incorporated byreference herein in their entirety, a sensitive method was developedthat is able to detect rare RT products in order to probe the structuresof low-abundance RNAs in living cells. This method increases thesensitivity of detection 100.000-fold over the conventional RT-basedmethod. It is demonstrated that both DMS and SHAPE chemistries permit invivo RNA structural probing in Arabidopsis thaliana, an important modelplant species and eukaryote. Notably, the in vivo SHAPE reagent,2-methylnicotinic acid imidazolide (NAI) (Spitale, R. C. et al., Nat.Chem. Biol. 9:18-20, 2013) was employed, and the first examples of invivo SHAPE probing in plants were presented. The RT-based method (FIG.25, first three steps) was used to successfully query the structures ofrRNA (25S rRNA and 5.8S rRNA) and chloroplast mRNA (PSBA) in A.thaliana. Then, a selective amplification strategy was developed toestablish a highly sensitive and robust method, ‘DMS/SHAPE-LMPCR’, whichuses ssDNA ligation, and achieves a 5-log enhancement in sensitivity.Using this LMPCR-based approach (FIG. 25, all five steps), DMS/SHAPEmodification signals were uncovered from low-abundance RNAs, and theirRNA structures are revealed for the first time in vivo.

As demonstrated herein, DMS/SHAPE-LMPCR, using ssDNA ligation, achievesattomole sensitivity, a 100.000-fold improvement over conventionalmethods. The structure of low-abundance U12 small nuclear RNA (snRNA) isprobed in Arabidopsis thaliana and in vivo evidence is providedsupporting the derived phylogenetic structure. Interestingly, incontrast to mammalian U12 snRNAs, the loop of the SLIIb in U12 snRNA isvariable among plant species, and DMS/SHAPE-LMPCR determines it to beunstructured. The effects of proteins on 25S rRNA, 5.8S rRNA and U12snRNA structure is revealed, illustrating the critical importance ofmapping RNA structure in vivo. The universally applicable method opensthe door to identifying and exploring the specific structure-functionrelationships of the multitude of low-abundance RNAs that prevail inliving cells.

The sensitivity limits of the standard in vivo RT-based assay were firstdetermined, using DMS probing reagent as an example. The total input RNAwas serially diluted until the observable DMS modification pattern for5.8S rRNA was lost. It was found that a relatively large amount of 5.8SrRNA (˜1 pmol) is necessary for conventional DMS-RT. This isapproximately the amount of 5.8S rRNA found in 2 μg of a ‘total RNA’extraction, which immediately presents a problem if one wants to assaythe many much lower abundance RNAs. Without a new approach an RNA at100,000-fold lower abundance than 5.8S rRNA would require ˜0.2 g oftotal RNA input for the DMS-RT assay, which is clearly impractical. Toimprove sensitivity, an amplification-based method was explored anddeveloped, which is referred to as ‘DMS/SHAPE-LMPCR’ (FIG. 25, allsteps). In this approach, a DNA adaptor is ligated to the 3′ end of thecomplementary DNA (cDNA), and the ligated cDNA is PCR amplified using agene-specific and an adaptor-specific primer. With this approach, it wasfound that the DMS modification pattern of 5.8S rRNA is observable evenat a 10-attomole (10⁻¹⁷) level of 5.8S rRNA input, which represents aremarkable 100,000-fold enhancement in sensitivity. Notably, themodification pattern derived from DMS-LMPCR was consistent with thepattern derived from the standard DMS-RT data, with strong Pearsoncorrelation coefficient (PCC) between the normalized DMS reactivitiesfor different regions of 5.8S rRNA ranging from 0.72 to 0.82.

Experiments were also conducted to compare the ssDNA ligation method ofthe invention to the prior Circligase-based ssDNA ligation method. Thesedata demonstrate that the ssDNA ligation method of the invention, usingT4 DNA ligase, provides more quantitative results in DMS-LMPCR thanCircligase-based ssDNA ligation, using Arabidopsis thaliana 5.8S rRNA asan example. For example, FIG. 26, depicts the results of comparisonexperiments, comparing the DMS-RT method (left panel, lane 6) withDMS-LMPCR using either T4 DNA ligase (left panel, lane 8) or CircligaseI (left panel, lane 10). The rectangles depict new bands observed onlyby the T4 DNA ligase method, which were not observed by the Circligasemethod. The gel on the right panel is a technical replicate forDMS/SHAPE-RT and DMS/SHAPE-LMPCR (using T4 DNA ligase) on 5.8S rRNA.

It is demonstrated herein, that ssDNA ligation may be used inDMS/SHAPE-LMPCR, which is a methodology that is demonstrated to increasethe sensitivity in determining the structure of low-abundancetranscripts. This demonstrates the utility of the ssDNA ligationcompositions and methods of the present invention in various protocols,including LMPCR.

Example 3 : In Vivo Genome-Wide Profiling of RNA Secondary StructureReveals Novel Regulatory Features

RNA structure has critical roles in processes ranging from ligandsensing to the regulation of translation, polyadenylation and splicing(Buratti et al., 2004, Mol Cell Biol, 24: 1387-1400; Cruz and Westhof,2009, Cell, 136: 604-609; Kozak, 2005, Gene, 361: 13-37; Sharp, 2009,Cell, 136: 577-580). However, a lack of genome-wide in vivo RNAstructural data has limited our understanding of how RNA structureregulates gene expression in living cells.

Most existing RNA structure mapping has been performed in vitro (Kerteszet al., 2010, Nature, 467: 103-107; Li et al., 2012, Plant Cell, 24:4346-4359; Zheng et al., 2010, PLoS Genet, 6: e1001141; Wan et al.,2012, Mol Cell, 48: 169-181). Among RNA structure probing reagents,dimethyl sulphate (DMS) can penetrate cells and has been used to mapstructures of high-abundance RNAs in vivo in various organisms (Senecoffand Meagher, 1992, Plant Mol Biol, 18: 219-234; Wells et al., 2000,Methods, Enzymol, 318: 479-493; Zuag and Cech, 1995, RNA, 1: 363-374;Zemora and Waldsich, 2010, RNA Biol, 7: 634-641). DMS methylates thebase-pairing faces of A and C of RNA in loops, bulges, mismatches andjoining regions. The base-pairing status of U and G nucleotides can beinferred from structural mapping of As and Cs, because constraining evensome nucleotides substantially improves predictions of other regions(Mathews et al., 2004, Proc Natl Acad Sci USA, 101: 7287-7292). However,a method for genome-wide study of RNA structure in vivo has beenlacking.

As demonstrated by Ding et al., (Ding et al., 2013, Nature, November 24.doi: 10.1038/nature12756), the contents of which are incorporated byreference herein in their entirety, a high-throughput, genome-wide invivo RNA structure probing method, Structure-Seq, was developed in whichdimethyl sulfate methylation of unprotected adenines and cytosines isidentified by next-generation sequencing. Application of this method toArabidopsis thaliana seedlings yielded the first in vivo genome-wide RNAstructure map at nucleotide resolution for any organism, withquantitative structural information across more than 10,000 transcripts.Here DMS methylation is combined with next-generation sequencing toestablish Structure-Seq, an in vivo quantitative measurement ofgenome-wide RNA secondary structure at nucleotide resolution.

Structure-Seq, which utilizes ssDNA ligation, is now described (FIG.27). While, the presently described experiments were conducted usingArabidopsis seedlings, Structure-Seq may be used to investigate anytissue or organism of interest. In the presently described experiments,Arabidopsis seedlings are treated with DMS. Reverse transcription isperformed using random hexamers (N₆) with adaptors (thicker blacklines). Reverse transcriptase stalls one nucleotide before DMS-modifiedAs and Cs (black crosses) (Zuag and Cech, 1995, RNA, 1: 363-374).Single-stranded (ss) DNA ligation attaches a single-stranded DNA linker(thicker black line) to the 3′ end. Double-stranded DNA is generated byPCR. A (−)DMS library is prepared in parallel. Deep sequencing isperformed with different indices for (+)DMS and (−)DMS libraries. Countsof the reverse transcriptase (RT) stops are normalized and subtracted.Pie charts depict percentages of RNA types for the (+)DMS (FIG. 27,left) and (−)DMS (FIG. 27, right) libraries. The non-rRNA and non-mRNAslice represent other RNA types plus unmappable reads

It was found that DMS-induced methylation sites were highly reproducible(Pearson correlation coefficient (PCC) of 0.91 for the two (+)DMSlibraries). Nucleotide modification in the (+)DMS library was specificto As and Cs. Notably, 98% of the combined 206 million sequence readswere mappable to the Arabidopsis genome. In this experiment, the readsinclude diverse classes of RNAs, with a predominance of mRNAs andribosomal RNAs. The reverse transcriptase stops are evenly distributedalong the transcripts, with no 3′ bias. In particular, 10,781transcripts had sufficient coverage at nucleotide resolution to obtainsecondary-structure constraints. Abundance of individual mRNAs inStructure-Seq correlated well with mRNA abundance from RNA-seq analyses(Oh et al, 2012, Nature Cell Biol, 14: 802-809).

It is demonstrated herein that ssDNA ligation can be used inStructure-Seq, a high-throughput genome-wide methodology that profilesRNA secondary structure with high accuracy and nucleotide resolution invivo. This demonstrates the utility of the ssDNA ligation compositionsand methods of the present invention in various high-throughputsequencing protocols, including Structure-Seq.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety. While this invention has been disclosed with referenceto specific embodiments, it is apparent that other embodiments andvariations of this invention may be devised by others skilled in the artwithout departing from the true spirit and scope of the invention. Theappended claims are intended to be construed to include all suchembodiments and equivalent variations.

What is claimed is:
 1. A method of producing a ligated single strandednucleic acid molecule, the method comprising: a) contacting asingle-stranded acceptor nucleic acid molecule with a population ofdonor nucleic acid molecules, wherein each of the donor nucleic acidmolecules comprises a stem region, and a single-stranded 3′ terminaloverhang region, wherein the sequence of the donor nucleic acid isdesigned to contain at least one mismatch internally in the stem region,and wherein at least the four 5′ terminal nucleotides of the donormolecule form Watson-Crick base pairs as part of the stem region, andwherein the donor molecule comprises at least one randomized templatingnucleotide in the 3′ terminal overhang region at the position nearestthe stem loop, thereby allowing unbiased hybridization with amultiplicity of different acceptor molecules; b) hybridizing the singlestranded 3′ terminal overhang region of the donor nucleic acid moleculeto the acceptor molecule under conditions where the stem structure isformed in the donor molecule, wherein the acceptor molecule may compriseany nucleotide sequence at the 3′ end, thereby forming an acceptor-donorhybrid molecule comprising a nick or gap between the acceptor nucleicacid and donor nucleic acid molecule; and c) ligating the 5′ end of thedonor nucleic acid molecule to the 3′ end of the acceptor nucleic acidmolecule with a ligase that is active under conditions where the stemstructure is formed in the donor nucleic acid molecule, therebygenerating a ligated product, whereby the yield of ligated product is atleast 65%.
 2. The method of claim 1, wherein the ligation in step c) isaccomplished after the hybridization in step b), wherein thehybridization step positions the acceptor and donor molecule in a waythat ligation occurs under conditions that allow for ligation betweenthe 5′ end of the donor nucleic acid molecule and the 3′ end of theacceptor nucleic acid molecule.
 3. The method of claim 1, wherein theligase comprises a DNA ligase.
 4. The method of claim 3, wherein the DNAligase is T4 DNA ligase.
 5. The method of claim 1, wherein the 3′terminal overhang region of donor molecule comprises at least 6 base(s).6. The method of claim 1, wherein the stem region of the donor moleculeis double stranded and comprises at least 6 nucleotide pairs.
 7. Themethod of claim 1, wherein the donor molecule further comprises a loopstructure, wherein the loop structure comprises at least 3 bases.
 8. Themethod of claim 7, wherein the loop structure comprises a portion of aprimer binding site.
 9. The method of claim 1, wherein at least onedonor molecule comprises a nucleotide sequence selected from the groupconsisting of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO:16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ IDNO: 21, SEQ ID NO: 22, SEQ ID NO: 28 and SEQ ID NO: 29, and wherein theligation is performed using T4 DNA ligase.
 10. The method of claim 1,wherein the stem of at least one donor molecule comprises a restrictionenzyme cleavage site.
 11. The method of claim 1, wherein at least onedonor molecule comprises at least six randomized templating nucleotidesin the 3′ overhang, and wherein the yield is at least 90%.
 12. Themethod of claim 11, wherein at least one donor molecule comprises thenucleotide sequence as set forth in SEQ ID NO: 29.